Protein metabolism

Protein turnover is the balance between protein synthesis and protein degradation. Proteins are naturally occurring polymers made up of repeating units of 20 different amino acids and range from small peptide hormones of 8 to 10 residues to very large multi-chain complexes of several thousand amino acids. Protein synthesis occurs on ribosomes - large intracellular structures consisting of a small subunit (33 proteins, 1900 nucleotides of ribosomal RNA) and a large subunit (46 proteins, 4980 nucleotides of rRNA) - that move along the messenger RNA (mRNA) copy of the gene (DNA) that was transcribed. The process of protein synthesis is called translation where the mRNA is read in triplets (codons), each triplet directing the addition of an amino acid (via its specific transfer RNA (tRNA)) to the growing polypeptide chain.

The assembly of new proteins requires a source of amino acids which come from either the proteolytic breakdown (digestion) of proteins in the gastrointestinal tract or the degradation of proteins within the cell. Intracellular protein degradation is done by proteolytic enzymes called proteases and occurs generally in two cellular locations - lysosomes and proteosomes. Lysosomal proteases digest proteins of extracellular origin that have been taken up by the process of endocytosis. Proteosomes, which are large, barrel-shaped, ATP-dependent protein complexes, digest damaged or unneeded intracellular proteins that have been marked for destruction by the covalent attachment of chains of a small protein, ubiquitin.

In contrast to the situation with glucose and fatty acids, amino acids in excess of those needed for biosynthesis cannot be stored and are not excreted. Rather, surplus amino acids are used as metabolic fuel. Most of the amino groups of surplus amino acids are converted into urea through the urea cycle, whereas their carbon skeletons are transformed into acetyl CoA, acetoacetyl CoA, pyruvate, or one of the intermediates of the tricarboxylic acid cycle. Hence, fatty acids, ketone bodies and glucose can be formed from amino acids.

Nature of amino acids and proteins

Proteins are naturally occurring polymers made up of repeating units of L-amino acids of which there are 20 – analogous to the 26 letters of the alphabet. Amino acids have the basic formula NH2-CHR-COOH and differ significantly in the chemical nature of their side chain ‘R’. They form peptide bond-linked (-CO-NH-) polymers[1] of the type ------NH-CHR1-CO-NH-CHR2-CO-NH-CHR3-CO-NH-CHR4-CO-NH-CHR5CO--------. Given the side chains ‘R’ can be large or small, charged or uncharged, hydrophilic or hydrophobic, chemically modifiable or unreactive, then the number and arrangement of these 20 ‘letters’ in the polypeptide chain governs both the size, surface chemistry and functional properties of the resulting protein.

Proteins can consist of just a single chain or of multiple chains held together by non-covalent and covalent bonds such as disulphide bonds (formed by the pairing and oxidation of cysteine residues). Some proteins like glucagon (single chain, 29 amino acids) and insulin (two chains, 51 amino acids) are small. In contrast some proteins like the insulin receptor are very large. The insulin receptor is a dimer, made up of two identical monomers of 1355 amino acid residues. Each monomer is proteolytically cleaved into an α-chain and a β-chain, with the four chain complex held together by two α- α and two α-β disulphide bonds.

Amino acids are categorized into two types - essential amino acids and non-essential amino acids. Essential amino acids are those which cannot be synthesized by the human body and thus have to be provided from the diet whereas non-essential amino acids are synthesized by the body. Essential amino acids include valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, threonine, lysine and histidine; whereas the non-essential amino acids are glycine, alanine, serine, asparagine, aspartic acid, glutamine, glutamic acid, proline, cysteine, tyrosine and arginine[2][3].

The proteins on degradation break down to individual amino acids. Depending on their metabolic fate amino acids are referred to as either glucogenic, ketogenic or glucogenic and ketogenic. The combination of protein catabolism and amino acid metabolism not only generates energy, but also generates key intermediates for the biosynthesis of certain non-essential amino acids, glucose and fat[2].

Protein synthesis

Figure 1. Steps in protein synthesis[4].  Click for enlarged view. Protein synthesis involves gene transcription (DNA to mRNA) in the nucleus and translation into polypeptide in the cytoplasm. Peptide bond synthesis occurs in the ribosome following codon (mRNA) / anti-codon (tRNA) base pairing. Source Ref [4].
Figure 1. Steps in protein synthesis[4]. Click for enlarged view. Protein synthesis involves gene transcription (DNA to mRNA) in the nucleus and translation into polypeptide in the cytoplasm. Peptide bond synthesis occurs in the ribosome following codon (mRNA) / anti-codon (tRNA) base pairing. Source Ref [4].
The overall process of protein synthesis extends from gene transcription in the nucleus to polypeptide synthesis on ribosomes in the cytoplasm and is summarized in Figure 1[4].

Genes and the genetic code.

The human genome contains approximately 20,000 protein-coding genes[5] that provide the instructions for the 250,000 to 1,000,000 proteins[6] that operate in the human body over its lifetime. The number of proteins exceeds the number of genes because one gene can code for more than one protein and many proteins exist in multiple forms due to post-synthetic chemical modifications. In addition there are thousands of non-coding RNA genes that help regulate the protein coding genes[5].

Figure 2. DNA structure showing the double helix and the base pairing (hydrogen bonds) where A pairs with T and G pairs with C. Source: Ref [7] Click for enlarged view.
Figure 2. DNA structure showing the double helix and the base pairing (hydrogen bonds) where A pairs with T and G pairs with C. Source: Ref [7] Click for enlarged view.
Genetic information is stored in the nucleus of cells in the form of deoxyribose nucleic acid (DNA). DNA is composed of just four building blocks (bases) adenine (A), guanine (G), cytosine (C) and thymine (T) linked by a deoxyribose -phosphate backbone to form a double helix (Figure 2)[7].

Figure 3. The genetic code. Click for enlarged view. The genetic sequence of DNA bases A, G, C, and T is copied and processed into the corresponding sequence of messenger RNA bases A, G, C and U for translation into polypeptide by the ribosome. The sequence of bases is read in triplets with sixty four possible combinations.  Methionine (Met) and tryptophan (Trp) are each coded for by a single triplet (codon), asparagine (Asn), aspartic acid (Asp), glutamine (Gln), glutamic acid (Glu), cysteine (Cys), phenylalanine (Phe), tyrosine (Tyr) and lysine (Lys) each have two codons; isoleucine (Ile) has three; glycine (Gly), alanine (Ala), valine (Val),  threonine (Thr) and proline (Pro) have four codons while serine (Ser) and arginine (Arg) have six.  There are three stop codons that denote the C-terminus of the translated protein. Source: Ref [8].
Figure 3. The genetic code. Click for enlarged view. The genetic sequence of DNA bases A, G, C, and T is copied and processed into the corresponding sequence of messenger RNA bases A, G, C and U for translation into polypeptide by the ribosome. The sequence of bases is read in triplets with sixty four possible combinations. Methionine (Met) and tryptophan (Trp) are each coded for by a single triplet (codon), asparagine (Asn), aspartic acid (Asp), glutamine (Gln), glutamic acid (Glu), cysteine (Cys), phenylalanine (Phe), tyrosine (Tyr) and lysine (Lys) each have two codons; isoleucine (Ile) has three; glycine (Gly), alanine (Ala), valine (Val), threonine (Thr) and proline (Pro) have four codons while serine (Ser) and arginine (Arg) have six. There are three stop codons that denote the C-terminus of the translated protein. Source: Ref [8].
As there are 20 amino acids, the code is not read as a single letter (4 possibilities only) or double letter (4x4 – 16 combinations) format, rather it is read in triplets called codons, with 4x4x4 (64) total combinations coding for all 20 amino acids as well as some punctuation (Stop/Start) instructions (Figure 3)[8].

Transcription

Proteins are not synthesized directly from DNA, but from an RNA (ribose nucleic acid) copy derived from one strand of DNA by a process called transcription[9]. Transcription occurs in the nucleus. The sections of the RNA gene copy that correspond to regions in the DNA that do not code for residues in the final protein (introns), are removed and the processed RNA copy - called messenger RNA - is transported to the cytoplasm where protein synthesis takes place. RNA contains three of the same bases as DNA, (adenine, guanine, cytosine) but employs uracil as the fourth base rather than thymine[9] (Figure 3). Gene transcription is controlled by special DNA-binding proteins called transcription factors[10] that are synthesized and activated/inactivated by protein hormones such as insulin and glucagon and non-protein hormones such as corticosteroids. Click to view movie of DNA transcription[11].

The role of Transfer RNA

A transfer RNA (tRNA) is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length. It serves as the physical link between the nucleotide sequence of the mRNA being translated and the resulting amino acid sequence of the synthesized protein. Thus tRNA is the means by which the correct amino acid - required to match each codon (triplet of bases) in the transcribed mRNA- is positioned in the peptidyl-transferase centre of the ribosome. One end of each tRNA contains a three-nucleotide sequence called the anticodon that can form three base pairs with a complementary three-nucleotide codon in mRNA during protein biosynthesis. Covalently attached to the other end of each tRNA is the amino acid that corresponds to the mRNA codon sequence. Each type of tRNA molecule can have only one type of amino acid attached to it and this is synthesized by enzymes called aminoacyl tRNA synthetases. One molecule of ATP is consumed in this process. Given the genetic code contains multiple codons that specify the same amino acid (Figure 3), there will be several tRNA molecules bearing different anticodons which also carry the same amino acid[12].

Translation on ribosomes

Protein synthesis is carried out in the cytoplasm by ribosomes - massive protein and RNA complexes that translate the nucleotide code on messenger RNA (mRNA) into functional protein[13][14][15]. Eukaryotic organisms, which include humans, have two ribosomal subunits, the large 60S and small 40S, which combine to form the functional 80S complex. In contrast, prokaryotes such as bacteria have similar, but smaller subunits — a large 50S and small 30S, which combine to form a 70S complex[13].

Ribosomes have been the focus of structural and biochemical studies for more than 50 years and in 2000, Tom Steitz’s laboratory at Yale University in Hartford Connecticut, published a high-resolution (2.4 Å) structure, of the large 50S subunit in the journal Science[14]. At this resolution, the researchers were able to definitively place nearly all of the 50S subunit’s 3,045 nucleotides and 31 proteins[13][14]. The structure revealed that the ribosome is a ribozyme because the catalytic peptidyl transferase activity that catalyses peptide bond formation, linking the amino acids together in the growing peptide chain, is performed by the ribosomal RNA[13][14]. Numerous initiation, elongation and release factors ensure that protein synthesis occurs progressively and with high specificity. In the past few years, high-resolution structures have provided molecular snapshots of different intermediates in ribosome-mediated translation in atomic detail[15]. Together, these studies have revolutionized our understanding of the mechanism of protein synthesis[15]. Tom Steitz shared the 2009 Nobel Prize for Chemistry with Venkatraman Ramakrishnan from the MRC Laboratory of Molecular Biology, Cambridge, United Kingdom, and Ada E. Yonath from the Weizmann Institute of Science, Rehovot, Israel ‘for their studies of the structure and function of the ribosome’ [16].

A detailed description of these discoveries can be found in the original papers and Tom Steitz’s review[15] and in the movies available at the following links ‘mRNA translation and protein synthesis[17] and ‘ribosome structure and function[18]. Only a brief summary will be provided here. The process of protein synthesis on ribosomes involves binding the mRNA in a tunnel formed between the two ribosomal subunits and initiating protein synthesis at the first codon. The two ribosomal subunits perform different roles in protein synthesis. The small ribosomal subunit mediates the correct inter¬actions between the anticodons of the tRNAs and the codons in the mRNA that they are translating in order to determine the order of the amino acids in the protein being synthesized. The large subunit contains the peptidyl-transferase centre (PTC), which catalyses the formation of peptide bonds in the growing polypeptide[15].

Both subunits contain three binding sites A, P and E, for tRNA molecules that are in three different functional states (Figure 4). The A site binds the aminoacyl-tRNA that is about to be incorporated into the growing polypeptide chain, the P site positions the peptidyl-tRNA (ie the tRNA with the growing peptide chain attached) and the E site is occupied by the deacylated tRNA before it dissociates from the ribosome (ie the tRNA after its attached peptide chain has been transferred (covalently linked) to the incoming amino acid on the aminoacyl tRNA)[15].

Figure 4. An overview of steps in protein synthesis. Click for enlarged view. mRNA translation is initiated with the binding of tRNAfmet to the P site (not shown). An incoming tRNA is delivered to the A site in complex with elongation factor (EF)-Tu–GTP. Correct codon–anticodon pairing activates the GTPase centre of the ribosome, which causes hydrolysis of GTP and release of the aminoacyl end of the tRNA from EF Tu. Binding of tRNA also induces conformational changes in ribosomal (r)RNA that optimally orientates the peptidyl-tRNA and aminoacyl-tRNA for the peptidyl-transferase reaction to occur, which involves the transfer of the peptide chain onto the A site tRNA. The ribosome must then shift in the 3′ mRNA direction so that it can decode the next mRNA codon. Translocation of the tRNAs and mRNA is facilitated by binding of the GTPase EF G, which causes the deacylated tRNA at the P site to move to the E site and the peptidyl-tRNA at the A site to move to the P site upon GTP hydrolysis. The ribosome is then ready for the next round of elongation. The deacylated tRNA in the E site is released on binding of the next aminoacyl-tRNA to the A site. Elongation ends when a stop codon is reached, which initiates the termination reaction that releases the polypeptide. Reprinted by permission from Macmillan Publishers Ltd: [Nature Reviews Molecular Cell Biology 9, 242-253 (March 2008) doi:10.1038/nrm2352].
Figure 4. An overview of steps in protein synthesis. Click for enlarged view. mRNA translation is initiated with the binding of tRNAfmet to the P site (not shown). An incoming tRNA is delivered to the A site in complex with elongation factor (EF)-Tu–GTP. Correct codon–anticodon pairing activates the GTPase centre of the ribosome, which causes hydrolysis of GTP and release of the aminoacyl end of the tRNA from EF Tu. Binding of tRNA also induces conformational changes in ribosomal (r)RNA that optimally orientates the peptidyl-tRNA and aminoacyl-tRNA for the peptidyl-transferase reaction to occur, which involves the transfer of the peptide chain onto the A site tRNA. The ribosome must then shift in the 3′ mRNA direction so that it can decode the next mRNA codon. Translocation of the tRNAs and mRNA is facilitated by binding of the GTPase EF G, which causes the deacylated tRNA at the P site to move to the E site and the peptidyl-tRNA at the A site to move to the P site upon GTP hydrolysis. The ribosome is then ready for the next round of elongation. The deacylated tRNA in the E site is released on binding of the next aminoacyl-tRNA to the A site. Elongation ends when a stop codon is reached, which initiates the termination reaction that releases the polypeptide. Reprinted by permission from Macmillan Publishers Ltd: [Nature Reviews Molecular Cell Biology 9, 242-253 (March 2008) doi:10.1038/nrm2352].

Of central interest are the mechanisms of peptide bond formation and mRNA decoding, which are crucial processes in the elongation phase of protein synthesis by the ribosome. During this phase of protein synthesis, nascent polypeptides are elongated from the N- to the C-terminus by the addition of one amino acid at a time. This process is facilitated by two protein factors: elongation factor Tu (EF-Tu), which facilitates the delivery of aminoacyl-tRNA to the A site of the ribosome, and elongation factor G (EF G), which promotes the translocation of the tRNAs and associated mRNA from their positions in the A and P sites to the P and E sites, respectively, and dissociates the previously bound E-site tRNA[15].

The accurate delivery of the correct aminoacyl-tRNA to the A site involves at least two distinct steps: (i) an interaction between the anticodon base triplet in the tRNA and the corresponding codon of the mRNA that resides in the A site of the ribosome and (ii) the communication of this correct formation of anticodon/codon Watson–Crick base pairing to the GTPase centre located in the large ribosomal subunit ~70 Å away, which results in the hydrolysis of the GTP bound to EF Tu. This GTP hydrolysis changes the conformation of EF-Tu resulting in its release from the tRNA and ribosome and the subsequent accommodation of the aminoacyl end of the tRNA into the peptidyl-transferase centre (PTC), which is followed rapidly by peptide bond formation[15].

At the end of the elongation cycle when the stop codon has been positioned in the A site, one of two protein release factors (RFs), RFI or RFII, binds to the A site and promotes the deacylation of the peptidyl-tRNA. A recycling factor, with the help of EF G, then leads to the dissociation of the release factor and the two ribosomal subunits[15].

Protein catabolism

The assembly of new proteins requires a source of amino acids. These building blocks are generated by the digestion of proteins in the gastrointestinal tract and the degradation of proteins within the cell.

Protein hydrolysis in the digestive tract

Protein digestion begins in the stomach, where the acidic environment favors protein denaturation. Denatured proteins are more accessible as substrates for proteolysis than are native proteins. The primary proteolytic enzyme of the stomach is the aspartate protease pepsin, a nonspecific protease that, remarkably, is maximally active at pH 2. Thus, pepsin can be active in the highly acidic environment of the stomach, even though other proteins undergo denaturation there. Protein degradation continues in the lumen of the intestine owing to the activity of proteolytic enzymes secreted by the pancreas. These are the serine proteases trypsin and chymotrypsin, and the carboxypeptidases (zinc metalloenzymes).

This battery of enzymes displays a wide array of specificity, and so the substrates are degraded into free amino acids as well as di- and tripeptides. Digestion is further enhanced by proteases, such as aminopeptidase N[19], Figure 5. Digestion and absorption of proteins. From Berg et al.,[20]. Click to enlarge
Figure 5. Digestion and absorption of proteins. From Berg et al.,[20]. Click to enlarge
that are located in the plasma membrane of the intestinal cells. Aminopeptidases digest proteins from the amino-terminal end. Single amino acids, as well as di- and tripeptides, are transported into the intestinal cells from the lumen and subsequently released into the blood for absorption by other tissues (Figure 5)[20].

Protein turnover and intracellular protein breakdown

Protein turnover—the degradation and resynthesis of proteins—takes place constantly in cells. Protein degradation is as essential to the cell as protein synthesis. It is required to supply amino acids for fresh protein synthesis; to remove excess enzymes and to remove transcription factors that are no longer needed[21]. Processes regulated by protein degradation include gene transcription, cell-cycle progression, organ formation, circadian rhythms, inflammatory responses, tumour suppression, cholesterol metabolism and antigen processing[20].

Although some proteins are very stable, many proteins are short lived, particularly those that are important in metabolic regulation. Altering the amounts of these proteins can rapidly change metabolic patterns. In addition, cells have mechanisms for detecting and removing damaged proteins. A significant proportion of newly synthesized protein molecules are defective because of errors in translation. Even proteins that are normal when first synthesized may undergo oxidative damage or be altered in other ways with the passage of time[20].

There are two major intracellular devices in which damaged or unneeded proteins are broken down: proteasomes and lysosomes[21].

Proteasomes deal primarily with endogenous proteins, ie, proteins that were synthesized within the cell such as:

  • transcription factors
  • cyclins (which must be destroyed to prepare for the next step in the cell cycle)[22]
  • proteins encoded by viruses and other intracellular pathogens
  • proteins that are folded incorrectly because of translation errors or because they are encoded by faulty genes (as in cystic fibrosis), or they have been damaged by other molecules in the cytosol[21].

Lysosomes deal primarily with

  • extracellular proteins, e.g., plasma proteins, that are taken into the cell, e.g., by endocytosis[23].
  • cell-surface membrane proteins that are used in receptor-mediated endocytosis[23].
  • proteins (and other macromolecules) engulfed by autophagosomes[24].

Proteasomal degradation of poly-ubiquitinated intracellular proteins

Figure 6. Cartoon of the proteasome[21]. It consists of a core particle made up of 2 copies of 14 different proteins, assembled into 2 rings of 7. The 4 rings are stacked on each other (like 4 doughnuts). There are two identical regulatory particles (RPs), one at each end of the core particle (CP). Each RP is made of 19 different proteins (none of them the same as those in the CP). Six of these are ATPases[21].
Figure 6. Cartoon of the proteasome[21]. It consists of a core particle made up of 2 copies of 14 different proteins, assembled into 2 rings of 7. The 4 rings are stacked on each other (like 4 doughnuts). There are two identical regulatory particles (RPs), one at each end of the core particle (CP). Each RP is made of 19 different proteins (none of them the same as those in the CP). Six of these are ATPases[21].
Damaged or unneeded proteins are marked for destruction by the covalent attachment of chains of a small protein, ubiquitin. Polyubiquitinated proteins are subsequently degraded by a large, ATP-dependent complex called the proteasome [20]. Proteasomes provide a controlled method for breaking down proteins safely within the environment of the cell. They chop obsolete or damaged proteins into small pieces, about 2 to 25 amino acids in length. Most of these are then completely broken down into amino acids by peptidases in the cell[25].

A simplified structure of the proteasome is shown in Figure 6.

The process of protein degradation in the proteasome involves the following stages.

Ubiquination. The proteasome is a multisubunit enzyme complex that plays a central role in the regulation of proteins that control cell-cycle progression and apoptosis, and has therefore become an important target for anticancer therapy. Before a protein is degraded, it is first flagged for destruction by the ubiquitin conjugation system, which ultimately results in the attachment of a polyubiquitin chain on the target protein[26]. Ubiquination involves three enzymes, designated E1, E2 and E3. Initially, the terminal carboxyl group of ubiquitin is joined in a thioester bond to a cysteine residue on E1 (Ubiquitin-Activating Enzyme). This is an ATP-dependent step. The ubiquitin is then transferred to a sulfhydryl group on E2 (Ubiquitin-Conjugating Enzyme). E3, a Ubiquitin-Protein Ligase then promotes transfer of ubiquitin from E2 to the ε-amino group of a lysine residue of the targeted protein that has been recognized by that E3, forming an isopeptide bond. There are many distinct Ubiquitin ligases with differing substrate specificity[27]. More ubiquitins may be added to form a chain of ubiquitins, the terminal carboxyl of each ubiquitin being linked to the ε-amino group of a lysine residue (Lys29 or Lys48) of the adjacent ubiquitin in the chain. A chain of four or more ubiquitin molecules targets proteins for degradation in proteasomes. Attachment of a single ubiquitin to a protein has other regulatory effects[27].

Binding and denaturation. The proteasome’s 19S regulatory cap at each end of the core, contains a large collection of regulatory subunits, (coloured blue in right-hand panel of Figure 7), that recognize proteins that are tagged with ubiquitin and queued up for destruction. Once bound the protein is unfolded and denatured by a set of ATPases (colored magenta in right-hand panel of Figure 7) using the energy of ATP. The unfolded protein is then translocated into the central cavity of the core particle[25].

Hydrolysis. Since proteasomes perform their job inside a cell, Figure 7. 3D structure of the proteasome. Click for enlarged view. Left panel is a ribbon diagram of the yeast 26S proteasome[27][28], right panel is a space filled model created by integrating several partial crystallographic structures into a near-atomic reconstruction of the proteasome obtained by analysis of 2.4 million images from electron microscopy[25].
Figure 7. 3D structure of the proteasome. Click for enlarged view. Left panel is a ribbon diagram of the yeast 26S proteasome[27][28], right panel is a space filled model created by integrating several partial crystallographic structures into a near-atomic reconstruction of the proteasome obtained by analysis of 2.4 million images from electron microscopy[25].
surrounded by proteins, the protein-cutting ability of proteasomes is carefully controlled. The active sites are hidden away inside a cylindrical "core" particle, shown here in yellow and red in right-hand panel of Figure 7[28]. The proteolytic core is composed of 2 inner β rings and 2 outer α rings. The 2 β rings each contain 3 proteolytic sites named for their trypsin-like, post-glutamyl peptide hydrolase-like (PGPH) (i.e., caspase-like), or chymotrypsin-like activity. These proteases on the inner surface of the two middle "doughnuts" break various specific peptide bonds of the denatured protein chain, producing a set of peptides averaging about 8 amino acids in length. These leave the core particle by an unknown route where they may be further broken down into individual amino acids by peptidases in the cytosol or in mammals may be incorporated in a class I histocompatibility molecule to be presented to the immune system as a potential antigen.

Ubiquitin release. As the denatured protein is hydrolysed the regulatory particle releases the pre-bound ubiquitins for reuse in the process[26].

Lysosomal proteolysis (endocytosed and organellar proteins)

Lysosomes contain a large variety of proteases that degrade proteins and other substances taken up by endocytosis[29]. Lysosomal proteases include cathepsins (cysteine proteases), aspartate proteases and one zinc protease. Materials taken into a cell by inward budding of vesicles from the plasma membrane may be processed first in an endosomal compartment and then delivered into the lumen of a lysosome by fusion of a transport vesicle[29]. Solute transporters embedded in the lysosomal membrane catalyze the exit of the products of lysosomal digestion (e.g., amino acids, sugars, cholesterol) to the cytosol. Lysosomes have a low internal pH due to activity of vacuolar ATPase, a H+ pump homologous to (but distinct from) the mitochondrial F1F0 ATPase[29] (see oxidative phosphorylation). All intra-lysosomal hydrolases exhibit acidic pH optima.

Caspases and apoptosis

Caspases are cysteine proteases involved in the activation and implementation of apoptosis (programmed cell death). Caspases get their name from the fact that they cleave on the carboxyl side of aspartic acid[30].

Caspases are a family of endoproteases that provide critical links in cell regulatory networks controlling inflammation and cell death. The activation of these enzymes is tightly controlled by their production as inactive zymogens that gain catalytic activity following signaling events promoting their aggregation into dimers or macromolecular complexes. Activation of apoptotic caspases results in inactivation or activation of substrates, and the generation of a cascade of signaling events permitting the controlled demolition of cellular components[30].

Activation of inflammatory caspases results in the production of active pro-inflammatory cytokines and the promotion of innate immune responses to various internal and external insults. Dysregulation of caspases underlies human diseases including cancer and inflammatory disorders, and major efforts to design better therapies for these diseases seek to understand how these enzymes work and how they can be controlled[30]. Apoptosis and caspase function is one of the most heavily researched fields in molecular medicine.

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