Question 1.
Sarin is a potent neurotoxin and novel chemical weapon that inhibits acetylcholinesterase (AChE) to cause rapid death by paralysis and asphyxiation. AChE is an important enzyme that clears the neuromuscular junction synaptic cleft of excess acetylcholine (ACh), the neurotransmitter responsible for muscle contraction. The nucleophilic serine of the AChE Glu-His-Ser catalytic triad hydrolyzes ACh to degrade the neurotransmitter. Sarin is an organophosphorus ester that binds to the serine residue hydroxyl group of the catalytic triad system of AChE. Sarin’s potency derives from oxygen’s high binding affinity for phosphorus. Sarin’s fluorinated phosphorus is electrophillically activated by fluorine’s large electronegativity, thereby promoting the nucleophilic substitution of AChE’s activated serine hydroxyl group.
The strength of the covalent bond between the enzyme’s serine and Sarin’s organophosphorus ester dominates the binding affinity of AChE to ACh. By this mechanism, Sarin binds to AChE’s active site and irreversibly inhibits the enzyme, allowing ACh to accumulate in the neuromuscular junction. Since ACh excites the postsynaptic neuron of the neuromuscular junction, Sarin’s activity prevents muscles (such as the diaphragm) from relaxing. Death by asphyxiation usually occurs within minutes of exposure and cures are typically ineffective due to the strength of the Sarin-AChE phosphoester linkage.
Organophosphorus chemistry has applications beyond chemical warfare, however. Sarin’s inhibitory mechanism combines both biochemical and synthetic concepts necessary to understand drug design and disease biochemistry. General organophosphorus compounds are not dissimilar to Sarin in their mechanisms and activity. In fact, much of medicinal chemistry relies on understanding the concepts behind Sarin’s mechanism of action and potency. Sarin binds to ACh’s catalytic triad similar to other organophosphate esters’ enzyme interactions. The catalytic triad is a cornerstone of many enzymatic pathways and, therefore, a prime target for drug inhibition. As contenders at the forefront of novel drug discovery, organophosphorus esters exhibit versatile biochemical properties to inhibit enzymes at the catalytic triad. Modifying the phosphorus functional residues of organophosphorus compounds varies their biological activities and half lives. Similarly, molecular accessories to the functional components around the phosphorus alter lipophilicity, enzyme binding properties, and structural features. Modern drug design uses these elements to design novel organophosphorus compounds able to inhibit certain enzymatic pathways unlike any other. Organophosphate enzyme inhibition encompasses enzyme kinetics, activity, the interdependence of structure and function, and the chemical mechanisms exploitable for drug development. Conclusively, these compounds uncover the potential of organophosphates in medicinal chemistry while implicating protein biochemistry. Sarin’s reactive mechanism exemplifies this relationship and the biological potential of organic phosphonates.
Question 2.
We briefly mentioned Huntington’s Disease (HD), the htt gene, and the Huntingtin protein, but never fully explored the protein biochemistry and pathology behind them. HD encompasses the symptoms of ALS, Parkinson’s, and Alzheimer’s alike; the genetics and biochemistry at play must be similarly robust. Expectably, HD pathology is not fully understood. We know the key protein, Huntingtin, plays a vital role in neuronal cellular maintenance, protein metabolism, and regulation. Researchers are still trying to pinpoint the function of Huntingtin, however, as they are trying to understand the effects of mutating Huntingtin-coding htt. There is a lot to learn about and challenge oneself with regarding HD and its biochemical mechanisms. Huntingtin, htt mutation effects, and HD exhibit features discussed throughout the course, making them a prime topic of potential debate.
Huntington’s Disease is a fatal genetic neurodegenerative disorder that drastically affects one’s physical and mental capacities. Appearing around age 40, symptoms include changes in personality, degraded cognition skills, slurred speech, loss of motor function, and dementia. The disease currently has lackluster treatments and no cure. Though the exact genetic and biochemical mechanisms behind HD continue to evade modern medicine, Huntingtin and the htt gene are known key factors in the disease.
Huntingtin is present throughout the body but concentrates in central nervous system neurons and especially in the brain. Huntingtin is a large (347 kDa) protein consisting of 3,144 amino acids with glutamate caps on either end coded for by CAG repeats in htt. HEAT repeats, Huntingtin’s primary functional regions, work as Elongation factors, protein 2A phosphatases, and TOR lipid kinases. Wild type Huntingtin maintains the proteostasis of neuronal proteins, modulates neuronal vesicle protein transport, and acts as an anti-apoptotic factor. Similarly, wt-Huntingtin associates with the cellular cytoskeleton and microtubules. The HEAT repeats coordinate these interactions.
The HEAT repeats must be sterically and electrostatically accessible for normal protein function. Expanded CAG repeats in mutant htt introduce excessive glutamate residues into Huntington. Glutamate’s size and length, polarity, and negative charge as an acidic amino acid interfere with HEAT-to-substrate interactions. Additional Glu on either end of Huntingtin also tangles the protein through electrostatic interference, further preventing the activity of the HEAT repeats. Expanded Glu repeats, therefore, inhibit the structure-to-function correlation of Huntingtin, prevent proper protein metabolism, and allow neuronal apoptosis, causing HD.
Question 3.
Reporter genes are protein-coding sequences inserted downstream from a response element gene. Response elements are activated by linked transcription factors. Pathway activity can be monitored by this coupling; reporter genes will be expressed in parallel to their response elements. Ubiquitination signalling triggers MID1 expression in response to protein-misfolding transcription factors. MID1 functions as a microtubule-associated Ubiquitin E3 ligase to degrade misfolded Protein Phosphatase 2A (PP2A). Misfolded PP2A activates MID1 expression. MID1 is the response element here. Transfecting cells with plasmid MID1-Luciferase DNA would cause simultaneous expression of both proteins during Ubiquitination activity. MID1-Luciferase expression will cause simultaneous luminescence. Thereover, Luciferase can be used to monitor the expression and activity of MID1 as an E3 ligase. In theory, Luciferase coupling could assay any protein expression by this mechanism.
GFP stands for green fluorescent protein because it fluoresces green when exposed to blue/ultraviolet light wavelengths. GFP functions by forming biologically stable chromophores of oxidized Serine, Tyrosine, and Glycine without any cofactors. This covalent complex can absorb light energy and re-emit light in the visible spectrum. We can use GFP’s fluorescence to analyze cellular gene expression by coupling GFP transcription to another protein such as MID1 in vivo. Downstream insertion of GFP after MID1 creates a green-fluorescent MID1-GFP complex. Expression of MID1-GFP will cause green fluorescence in MID1 concentrated cellular regions. Superimposing a Congo-red stained cellular component image over the green MID1-GFP image causes yellow regions to appear indicative of MID1 localization.
Question 4.
G-Protein Coupled Receptor
Cyclic AMP (cAMP), Inositol Triphosphate (IP3), Calcium Cations (Ca+2)
The GTP-bound G⍺1 subunit inhibits the function of Adenylate Cyclase. This prevents the conversion of ATP to cAMP and reduces the overall amount of cAMP.
The synthetic PEST-encoding sequence encourages proteolysis by signalling for protein degradation. Therefore, the PEST sequence reduces the biological half-life of the protein. A lower biological half-life increases protein turnover and, in turn, increases the sensitivity of reporter assays. With lower half-life, the PEST-modified luciferase has a more rapid response activity than the wild-type protein. A rapid response sensitivity allows researchers to detect short-term/rapid changes in gene expression. This way, leftover luciferase from other response elements does not interfere with detecting other pathway activities.
Yes, it can be determined exactly which pathway was activated.
The G⍺q-PLC subunit activates PIP2 which can activate both the NFAT-RE-terminal or SRE-terminal pathways. Both of these response elements invoke the transcription of the luciferase gene. This is an experimental problem. It is possible, however, to eliminate either the SRE or NFAT-RE response elements in culture, preventing the G⍺q pathway from activating two different response elements. Comparing SRE and NFAT knockout cultures can distinguish which pathway a drug is activating. Therefore, it is possible to determine whether the G⍺q-PLC or Gβ2 subunit was activated to trigger luciferase production.
Culture a group of cells without any SRE, only NFAT-RE response elements. Separately, culture a group of cells without any NFAT-RE, only SRE response elements. Knocking out the SRE response element will eliminate the possibility of triggering luciferase production through the GβƔ → Raf → ERK1/2 → SRE pathway. Knocking out the NFAT-RE response element will eliminate the possibility of triggering luciferase production through the G⍺q-PLC → PIP2 → DAG → PKC → Raf → SRE pathway. Without SRE, the G⍺q-PLC can activate only the NFAT-RE pathway. Without NFAT-RE, the G⍺q-PLC can still trigger luciferase production by activating SRE. If both cultures luminesce, that means G⍺q-PLC is activating NFAT in the SRE knockout culture and SRE in the NFAT culture, indicating the drug is activating G⍺q-PLC. If only the NFAT knockout culture luminesces, that means GβƔ is activating SRE in the NFAT knockout culture but cannot activate NFAT in the SRE knockout (because its pathway is not link to NFAT activation), indicating the drug is activating GβƔ. This method determines whether a drug activates the G⍺q-PLC or GβƔ pathway.