Substrate presentation

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Substrate presentation; A substrate (purple rectangle) is shown sequestered into a lipid domain (green lipids). The substrate's translocation to the disordered region (grey lipids) presents it to its enzyme (blue oval) where it is hydrolyzed. Substrate translocation.png
Substrate presentation; A substrate (purple rectangle) is shown sequestered into a lipid domain (green lipids). The substrate's translocation to the disordered region (grey lipids) presents it to its enzyme (blue oval) where it is hydrolyzed.

Substrate presentation is a biological process that activates a protein. The protein is sequestered away from its substrate and then activated by release and exposure of the protein to its substrate. [1] [2] A substrate is typically the substance on which an enzyme acts but can also be a protein surface to which a ligand binds. The substrate is the material acted upon. In the case of an interaction with an enzyme, the protein or organic substrate typically changes chemical form. Substrate presentation differs from allosteric regulation in that the enzyme need not change its conformation to begin catalysis. Substrate presentation is best described for nanoscopic distances (<100 nm). [3]

Contents

Examples

Amyloid precursor protein

Amyloid precursor protein (APP) is cleaved by beta and gamma secretase to yield a 40-42 amino acid peptide responsible for amyloid plaques associated with Alzheimer's disease. The secretase enzymes are regulated by substrate presentation. [4] The substrate APP is palmitoylated and moves in and out of GM1 lipid rafts in response to astrocyte cholesterol. Cholesterol delivered by apolipoprotein E (ApoE) drives APP to associate with GM1 lipid rafts. When cholesterol is low, the protein traffics to the disordered region and is cleaved by alpha secretase to produce a non-amylogenic product. The enzymes do not appear to respond to cholesterol, only the substrate moves.

Hydrophobicity drives the partitioning of molecules. In the cell, this gives rise to compartmentalization within the cell and within cell membranes. For lipid rafts, palmitoylation regulates raft affinity for the majority of integral raft proteins. [5] Raft regulation is regulated by cholesterol signaling.

Phospholipase D2

(PLD2) is a well-defined example of an enzyme activated by substrate presentation. [6] The enzyme is palmitoylated causing the enzyme to traffic to GM1 lipid domains or "lipid rafts". The substrate of phospholipase D is phosphatidylcholine (PC) which is unsaturated and is of low abundance in lipid rafts. PC localizes to the disordered region of the cell along with the polyunsaturated lipid phosphatidylinositol 4,5-bisphosphate (PIP2). PLD2 has a PIP2 binding domain. When PIP2 concentration in the membrane increases, PLD2 leaves the GM1 domains and associates with PIP2 domains where it then gains access to its substrate PC and commences catalysis based on substrate presentation. Presumably, the enzyme is capable of catalyzing a reaction in a lipid raft but lacks a substrate for activity.

Enzyme translocation; PLD (blue oval) is sequestered into cholesterol-dependent lipid domains (green lipids) by palmitoylation. PLD also binds PIP2(red hexagon) domains (grey shading) located separate from GM1 clusters in the plasma membrane and near phosphatidylcholine (PC). When PIP2 increases in the cell PLD translocates to PIP2 where it is exposed to and hydrolyzes PC to phosphatidic acid (red spherical lipid). Enzyme translocation.png
Enzyme translocation; PLD (blue oval) is sequestered into cholesterol-dependent lipid domains (green lipids) by palmitoylation. PLD also binds PIP2(red hexagon) domains (grey shading) located separate from GM1 clusters in the plasma membrane and near phosphatidylcholine (PC). When PIP2 increases in the cell PLD translocates to PIP2 where it is exposed to and hydrolyzes PC to phosphatidic acid (red spherical lipid).

Inflammation

(ADAM17), also called TACE, is sequestered into lipid rafts away from its substrate, membrane bound tumor necrosis factor (mTNF). [7] Cholesterol causes mTNF to cluster with ADAM17 in lipid rafts and shed soluble TNF (sTNF) which is an inflammatory cytokine.

Kinase Signaling

Receptor Tyrosine Kinases are cell surface receptors that bind to various polypeptide growth factors, cytokines, and hormones. Activation of RTKs is driven by palmitoylation and dimerization, a process facilitated by cholesterol within lipid rafts. [8] [9] Once dimerized, the receptor undergoes autophosphorylation, which triggers a subsequent phosphorylation cascade. This is a specific case where the substrate and the enzyme are the same molecule.


Protein Kinase C (PKC) is a class of enzymes that phosphorylates proteins. Its substrates are typically on the membrane surface where the enzyme is recruited by the lipid diacylglycerol. Thus a portion of PKC activation is through substrate presentation, i.e., by localization with its substrate on the membrane.

SARS-CoV-2

(Furin) (producing cell, replication). When cells are loaded with cholesterol furin traffics to GM1 lipid rafts where it is localized with the palmitoylated spike protein of SARS-CoV-2 and primes it for viral entry. [10]

(ACE2) (target Cell, viral entry), the receptor for SARS-CoV-2 ACE2 traffics SARS-CoV-2 to GM1 lipid rafts where it is endocytosed and exposed to cathepsin for cleavage and optimal cells fusion. [11] [12] In low cholesterol ACE2 traffics the virus to TMPRSS2 which also cleaves and allows viral entry but through a putative surface mechanism that is much less efficient. The sensitivity of ACE2 to cholesterol is thought to contribute to less severe COVID19 symptoms in children.

Mechanisms of activation

Sequestration

Sequestration is the process of moving a molecule to a lipid raft. Within the plasma membrane, sequestration is primarily driven by packing of saturated lipid with cholesterol or phase separation at very small distances (< 100 nm). At a macroscopic level, organelles and vesicle can limit access of an enzyme with to substrate.

Sequestration can both elevate and reduce the concentration of a protein in proximity to its substrate. When the substrate is present within a lipid raft, sequestration leads to an increased concentration of the protein near the substrate. Conversely, if the substrate is excluded from a lipid raft, sequestration results in decreased interaction between the protein and the substrate, as seen with PLD2.

Either the substrate of the enzyme can move. Movement is typically the disruption of palmitate mediated localization or organelle trafficking. For proteins that are both palmitoylated and bind PIP2, increasing the concentration of PIP2 favors trafficking of the enzyme out of lipid rafts to PIP2. PIP2 is primarily polyunsaturated which causes the lipid to localize away from lipid rafts and allows the PIP2 to oppose palmitate mediated localization. [13]

Regulation

Cholesterol

Cholesterol and polyunsaturated fatty acids (PUFAs) regulate lipid raft formation, hence the biological function of rafts. When saturated lipids and cholesterol increase in the membrane, lipid rafts increase their affinity for palmitoylated proteins. [14] PUFAs have the opposite effect, they fluidize the membrane.

PUFAs

PUFAs may also increase the concentration of signaling lipids. The arachidonic acid, a very common PUFA in the brain, incorporates into PC and PIP2. [15] Arachidonyl PC is a preferred substrate of PLD likely increasing the amount of PA in a cell. Regulation of raft function by cholesterol effectively regulates substrate presentation and the many palmitoylated proteins that utilize substrate presentation as a mechanism of activation. While speculative, the profound effect of cholesterol and PUFAs on human health is likely through physiological regulation of lipid raft function in cells.

Role in biology

Mechanosensation

Mechanical force (shear or swell) can independently disrupt the packing and resultant affinity of palmitate to lipid rafts. This disruption also causes PLD2 to favor trafficking to PIP2 domains. [16] The mechanosensitive ion channel TREK-1 is released from cholesterol dependent lipid rafts in response to mechanical force. This has the effect of dampening pain. [17]

Anaesthesia

Membrane-mediated anesthesia employs substrate presentation. General anesthetics propofol and inhaled anesthetics xenon, chloroform, isoflurane, diethyl ether disrupt lipid raft function and palmitate mediated localization of PLD2 to lipid rafts. [18] [19] Activation of PLD then activates TREK-1 channels. The membrane mediated PLD2 activation could be transferred to an anesthetic insensitive homolog TRAAK, rending the channel anesthetic sensitive.

Related Research Articles

<span class="mw-page-title-main">Cholesterol</span> Sterol biosynthesized by all animal cells

Cholesterol is the principal sterol of all higher animals, distributed in body tissues, especially the brain and spinal cord, and in animal fats and oils.

<span class="mw-page-title-main">Lipid-anchored protein</span> Membrane protein

Lipid-anchored proteins are proteins located on the surface of the cell membrane that are covalently attached to lipids embedded within the cell membrane. These proteins insert and assume a place in the bilayer structure of the membrane alongside the similar fatty acid tails. The lipid-anchored protein can be located on either side of the cell membrane. Thus, the lipid serves to anchor the protein to the cell membrane. They are a type of proteolipids.

<span class="mw-page-title-main">Theories of general anaesthetic action</span> How drugs induce reversible suppression of consciousness

A general anaesthetic is a drug that brings about a reversible loss of consciousness. These drugs are generally administered by an anaesthetist/anesthesiologist to induce or maintain general anaesthesia to facilitate surgery.

<span class="mw-page-title-main">Lipid raft</span> Combination in the membranes of cells

The plasma membranes of cells contain combinations of glycosphingolipids, cholesterol and protein receptors organised in glycolipoprotein lipid microdomains termed lipid rafts. Their existence in cellular membranes remains controversial. Indeed, Kervin and Overduin imply that lipid rafts are misconstrued protein islands, which they propose form through a proteolipid code. Nonetheless, it has been proposed that they are specialized membrane microdomains which compartmentalize cellular processes by serving as organising centers for the assembly of signaling molecules, allowing a closer interaction of protein receptors and their effectors to promote kinetically favorable interactions necessary for the signal transduction. Lipid rafts influence membrane fluidity and membrane protein trafficking, thereby regulating neurotransmission and receptor trafficking. Lipid rafts are more ordered and tightly packed than the surrounding bilayer, but float freely within the membrane bilayer. Although more common in the cell membrane, lipid rafts have also been reported in other parts of the cell, such as the Golgi apparatus and lysosomes.

Phosphatidic acids are anionic phospholipids important to cell signaling and direct activation of lipid-gated ion channels. Hydrolysis of phosphatidic acid gives rise to one molecule each of glycerol and phosphoric acid and two molecules of fatty acids. They constitute about 0.25% of phospholipids in the bilayer.

<span class="mw-page-title-main">Myristoylation</span>

Myristoylation is a lipidation modification where a myristoyl group, derived from myristic acid, is covalently attached by an amide bond to the alpha-amino group of an N-terminal glycine residue. Myristic acid is a 14-carbon saturated fatty acid (14:0) with the systematic name of n-tetradecanoic acid. This modification can be added either co-translationally or post-translationally. N-myristoyltransferase (NMT) catalyzes the myristic acid addition reaction in the cytoplasm of cells. This lipidation event is the most common type of fatty acylation and is present in many organisms, including animals, plants, fungi, protozoans and viruses. Myristoylation allows for weak protein–protein and protein–lipid interactions and plays an essential role in membrane targeting, protein–protein interactions and functions widely in a variety of signal transduction pathways.

<span class="mw-page-title-main">Phosphoinositide phospholipase C</span>

Phosphoinositide phospholipase C is a family of eukaryotic intracellular enzymes that play an important role in signal transduction processes. These enzymes belong to a larger superfamily of Phospholipase C. Other families of phospholipase C enzymes have been identified in bacteria and trypanosomes. Phospholipases C are phosphodiesterases.

<span class="mw-page-title-main">Phosphatidylinositol 4,5-bisphosphate</span> Chemical compound

Phosphatidylinositol 4,5-bisphosphate or PtdIns(4,5)P2, also known simply as PIP2 or PI(4,5)P2, is a minor phospholipid component of cell membranes. PtdIns(4,5)P2 is enriched at the plasma membrane where it is a substrate for a number of important signaling proteins. PIP2 also forms lipid clusters that sort proteins.

<span class="mw-page-title-main">Lipid signaling</span> Biological signaling using lipid molecules

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<span class="mw-page-title-main">Palmitoylation</span>

Palmitoylation is the covalent attachment of fatty acids, such as palmitic acid, to cysteine (S-palmitoylation) and less frequently to serine and threonine (O-palmitoylation) residues of proteins, which are typically membrane proteins. The precise function of palmitoylation depends on the particular protein being considered. Palmitoylation enhances the hydrophobicity of proteins and contributes to their membrane association. Palmitoylation also appears to play a significant role in subcellular trafficking of proteins between membrane compartments, as well as in modulating protein–protein interactions. In contrast to prenylation and myristoylation, palmitoylation is usually reversible (because the bond between palmitic acid and protein is often a thioester bond). The reverse reaction in mammalian cells is catalyzed by acyl-protein thioesterases (APTs) in the cytosol and palmitoyl protein thioesterases in lysosomes. Because palmitoylation is a dynamic, post-translational process, it is believed to be employed by the cell to alter the subcellular localization, protein–protein interactions, or binding capacities of a protein.

<span class="mw-page-title-main">PLD2</span> Protein-coding gene in the species Homo sapiens

Phospholipase D2 is an enzyme that in humans is encoded by the PLD2 gene.

<span class="mw-page-title-main">Phospholipase C</span> Class of enzymes

Phospholipase C (PLC) is a class of membrane-associated enzymes that cleave phospholipids just before the phosphate group (see figure). It is most commonly taken to be synonymous with the human forms of this enzyme, which play an important role in eukaryotic cell physiology, in particular signal transduction pathways. Phospholipase C's role in signal transduction is its cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), which serve as second messengers. Activators of each PLC vary, but typically include heterotrimeric G protein subunits, protein tyrosine kinases, small G proteins, Ca2+, and phospholipids.

<span class="mw-page-title-main">KCNK2</span> Protein-coding gene in the species Homo sapiens

Potassium channel subfamily K member 2, also known as TREK-1, is a protein that in humans is encoded by the KCNK2 gene.

<span class="mw-page-title-main">Acyl-protein thioesterase</span> Enzymes that cleave off lipid modifications on proteins

Acyl-protein thioesterases are enzymes that cleave off lipid modifications on proteins, located on the sulfur atom of cysteine residues linked via a thioester bond. Acyl-protein thioesterases are part of the α/β hydrolase superfamily of proteins and have a conserved catalytic triad. For that reason, acyl-protein thioesterases are also able to hydrolyze oxygen-linked ester bonds.

<span class="mw-page-title-main">Lipid-gated ion channels</span> Type of ion channel transmembrane protein

Lipid-gated ion channels are a class of ion channels whose conductance of ions through the membrane depends directly on lipids. Classically the lipids are membrane resident anionic signaling lipids that bind to the transmembrane domain on the inner leaflet of the plasma membrane with properties of a classic ligand. Other classes of lipid-gated channels include the mechanosensitive ion channels that respond to lipid tension, thickness, and hydrophobic mismatch. A lipid ligand differs from a lipid cofactor in that a ligand derives its function by dissociating from the channel while a cofactor typically derives its function by remaining bound.

Palmitate mediated localization is a biological process that trafficks a palmitoylated protein to ordered lipid domains.

<span class="mw-page-title-main">Cholesterol signaling</span>

Cholesterol is a cell signaling molecule that is highly regulated in eukaryotic cell membranes. In human health, its effects are most notable in inflammation, metabolic syndrome, and neurodegeneration. At the molecular level, cholesterol primarily signals by regulating clustering of saturated lipids and proteins that depend on clustering for their regulation.

PIP2 domains are a type of cholesterol-independent lipid domain formed from phosphatidylinositol and positively charged proteins in the plasma membrane. They tend to inhibit GM1 lipid raft function.

Membrane-mediated anesthesia or anaesthesia (UK) is a mechanism of action that involves an anesthetic exerting its effects through the lipid membrane. Established mechanism exists for both general and local anesthetics. The anesthetic binding site is within ordered lipids and binding disrupts the function of the ordered lipid. See Theories of general anaesthetic action for a broader discussion of purely theoretical mechanisms.

References

  1. ↑ Petersen, EN; Pavel, MA; Wang, H; Hansen, SB (28 October 2019). "Disruption of palmitate-mediated localization; a shared pathway of force and anesthetic activation of TREK-1 channels". Biochimica et Biophysica Acta (BBA) - Biomembranes. 1862 (1): 183091. doi: 10.1016/j.bbamem.2019.183091 . PMC   6907892 . PMID   31672538.
  2. ↑ Robinson, CV; Rohacs, T; Hansen, SB (September 2019). "Tools for Understanding Nanoscale Lipid Regulation of Ion Channels". Trends in Biochemical Sciences. 44 (9): 795–806. doi:10.1016/j.tibs.2019.04.001. PMC   6729126 . PMID   31060927.
  3. ↑ Yuan, Zixuan; Hansen, Scott B. (20 February 2023). "Cholesterol Regulation of Membrane Proteins Revealed by Two-Color Super-Resolution Imaging". Membranes. 13 (2): 250. doi: 10.3390/membranes13020250 . PMC   9966874 . PMID   36837753.
  4. ↑ Wang, Hao; Kulas, Joshua A.; Wang, Chao; Holtzman, David M.; Ferris, Heather A.; Hansen, Scott B. (17 August 2021). "Regulation of beta-amyloid production in neurons by astrocyte-derived cholesterol". Proceedings of the National Academy of Sciences. 118 (33): e2102191118. Bibcode:2021PNAS..11802191W. doi: 10.1073/pnas.2102191118 . ISSN   0027-8424. PMC   8379952 . PMID   34385305.
  5. ↑ Levental, I; Lingwood, D; Grzybek, M; Coskun, U; Simons, K (21 December 2010). "Palmitoylation regulates raft affinity for the majority of integral raft proteins". Proceedings of the National Academy of Sciences of the United States of America. 107 (51): 22050–4. Bibcode:2010PNAS..10722050L. doi: 10.1073/pnas.1016184107 . PMC   3009825 . PMID   21131568.
  6. ↑ Petersen, EN; Chung, HW; Nayebosadri, A; Hansen, SB (15 December 2016). "Kinetic disruption of lipid rafts is a mechanosensor for phospholipase D." Nature Communications. 7: 13873. Bibcode:2016NatCo...713873P. doi:10.1038/ncomms13873. PMC   5171650 . PMID   27976674.
  7. ↑ Tellier, Edwige; Canault, Matthias; Rebsomen, Laure; Bonardo, Bernadette; Juhan-Vague, Irène; Nalbone, Gilles; Peiretti, Franck (10 December 2006). "The shedding activity of ADAM17 is sequestered in lipid rafts". Experimental Cell Research. 312 (20): 3969–3980. doi:10.1016/j.yexcr.2006.08.027. PMID   17010968.
  8. ↑ Pike, LJ (30 December 2005). "Growth factor receptors, lipid rafts and caveolae: an evolving story". Biochimica et biophysica acta. 1746 (3): 260–73. doi: 10.1016/j.bbamcr.2005.05.005 . PMID   15951036.
  9. ↑ Paige, LA; Nadler, MJ; Harrison, ML; Cassady, JM; Geahlen, RL (25 April 1993). "Reversible palmitoylation of the protein-tyrosine kinase p56lck". The Journal of biological chemistry. 268 (12): 8669–74. PMID   8473310.
  10. ↑ Wang, Hao; Yuan, Zixuan; Pavel, Mahmud Arif; Hansen, Scott B. (29 May 2020). "The role of high cholesterol in age-related COVID19 lethality". bioRxiv: 2020.05.09.086249. doi:10.1101/2020.05.09.086249. PMC   7263494 . PMID   32511366.
  11. ↑ Hansen, Scott B.; Yuan, Zixuan (March 2023). "Getting in on the action: New tools to see SARS-CoV-2 infect a cell". Cell Chemical Biology. 30 (3): 233–234. doi:10.1016/j.chembiol.2023.02.010. PMC   10018748 . PMID   36931249.
  12. ↑ Wang, Hao; Yuan, Zixuan; Pavel, Mahmud Arif; Hansen, Scott B. (29 May 2020). "The role of high cholesterol in age-related COVID19 lethality". bioRxiv: 2020.05.09.086249. doi:10.1101/2020.05.09.086249. PMC   7263494 . PMID   32511366.
  13. ↑ Hansen, SB (May 2015). "Lipid agonism: The PIP2 paradigm of ligand-gated ion channels". Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1851 (5): 620–8. doi:10.1016/j.bbalip.2015.01.011. PMC   4540326 . PMID   25633344.
  14. ↑ Levental, I; Lingwood, D; Grzybek, M; Coskun, U; Simons, K (21 December 2010). "Palmitoylation regulates raft affinity for the majority of integral raft proteins". Proceedings of the National Academy of Sciences of the United States of America. 107 (51): 22050–4. Bibcode:2010PNAS..10722050L. doi: 10.1073/pnas.1016184107 . PMC   3009825 . PMID   21131568.
  15. ↑ Petersen, E. Nicholas; Gudheti, Manasa; Pavel, Mahmud Arif; Murphy, Keith R.; Ja, William W.; Jorgensen, Erik M.; Hansen, Scott B. (5 September 2019). "Phospholipase D Transduces Force to TREK-1 Channels in a Biological Membrane". bioRxiv   10.1101/758896 .
  16. ↑ Petersen, EN; Pavel, MA; Wang, H; Hansen, SB (28 October 2019). "Disruption of palmitate-mediated localization; a shared pathway of force and anesthetic activation of TREK-1 channels". Biochimica et Biophysica Acta (BBA) - Biomembranes. 1862 (1): 183091. doi: 10.1016/j.bbamem.2019.183091 . PMC   6907892 . PMID   31672538.
  17. ↑ Petersen, E. Nicholas; Pavel, Mahmud Arif; Hansen, Samuel S.; Gudheti, Manasa; Wang, Hao; Yuan, Zixuan; Murphy, Keith R.; Ja, William; Ferris, Heather A.; Jorgensen, Erik; Hansen, Scott B. (26 February 2024). "Mechanical activation of TWIK-related potassium channel by nanoscopic movement and rapid second messenger signaling". eLife. 12: RP89465. doi: 10.7554/eLife.89465 . PMC   10942622 . PMID   38407149.
  18. ↑ Petersen, EN; Pavel, MA; Wang, H; Hansen, SB (1 January 2020). "Disruption of palmitate-mediated localization; a shared pathway of force and anesthetic activation of TREK-1 channels". Biochimica et Biophysica Acta (BBA) - Biomembranes. 1862 (1): 183091. doi: 10.1016/j.bbamem.2019.183091 . PMC   6907892 . PMID   31672538.
  19. ↑ Pavel, Mahmud Arif; Petersen, E. Nicholas; Wang, Hao; Lerner, Richard A.; Hansen, Scott B. (19 June 2019). "Studies on the mechanism of membrane mediated general anesthesia". bioRxiv   10.1101/313973 .
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