Biological Interest HIV

Progress Report 1 1 Biological Interest: HIV ● A sexually transmitted disease, which alters immunodeficiency of the host. ● Viral infection through various ways. Unprotected sexual intercourse, shared needle or syringes, mother-to child transmission upon child birth or breastfeeding(HIV- positive mother), receiving contaminated blood, organ transplant from infected donor2 ● The HIV has a lipid envelope which is derived from the host cell membrane which contains glycoproteins(gp120 and gp41) on the surface. ● HIV targets immune cells, such as, dendritic, macrophage, and CD4+T Cells(T-helper cell). ● Like other invasive elements, HIV also infects through fusion of membrane ● HIV, through binding onto the receptor of the host cell, will initiate fusion. Here, the interaction between Gp 120 of HIV and CD4 of the host cell will bind HIV and the host cell.3 ● Upon fusion, an invasive element can now interfere with the host cell’s genome sequences. ● Once inside the host cell, the viral RNA is reverse transcribed into DNA, integrated and interrupting the host cell genome to produce viral organs for the next generation. ● HIV weakens the immune system by reducing the number of functional CD4+T cells. HIV Protein of interest: GP120/GP414 ● GP120 initiates binding to the receptors on the host cell. The linkage with GP120 and CD4 will trigger GP41 to undergo structural rearrangement, so the hydrophobic region will be on external side ● The GP41 will insert into the host cells membrane, promoting fusion between viral membrane and host membrane. ● Through membrane fusion, it will allow the viral genome to enter the host cell’s genome. ● One of the ways to neutralize the effect of GP120 and GP41 is to use fusion inhibitors, such as Enfivirocytes. ● Enfivirocytes is a competitive inhibitor which mimics the GP41 which promotes the fusion of the membrane. Note that modification of GP41 and binding of GP120 with CD4+T cell are still going to happen.5 Proposal A. To inhibit entry of the viral genome via fusion inhibitors, such as Fibrocyte. Determine specific mechanism of inhibition on fission. B. Determine mechanism upon structural change on gp 41. C. Determine interaction between gp41 and host membrane Biological Interest: Parkinson’s Disease6 ● Parkinson’s disease (PD) is a neurodegenerative disorder caused by the loss of dopaminergic neurons. ● There has been a correlation between brain inflammation being a contributing factor in the development of Parkinson's disease (PD). ● This shows a connection between activated microglia and the loss of dopaminergic neurons. ● When microglia undergo certain inflammatory stimuli, they activate and release inflammatory mediators such as mitogen-activated protein kinases (MAPKs). ● When the MAPK’s get phosphorylated, they turn into phosphorylated MAPK’s (p-MAPK’s). ● The p-MAPK’s induces a protein called inducible nitric oxide synthase (iNOS). ● iNOS catalyzes the production of nitric oxide (NO). ● NO is a very potent pro-inflammatory mediator. ● It has been shown that excessive expression of iNOS has been linked to various inflammatory conditions.7 ● It is crucial to emphasize that MAPKs (p38) play a significant role in regulating the production and secretion of pro-inflammatory substances by activated microglia.8 Parkinson’s Disease Protein of Interest 1: iNOS9 ● inducible nitric oxide synthase (iNOS) is an enzyme that is essential in the production of nitric oxide (NO) ● iNOS makes the amino acid L-arginine undergo oxidative deamination, forming NO.10 ● iNOS plays a crucial role in physiological processes such as blood pressure regulation and inflammation. ● iNOS is used as a marker in certain therapeutic instances. Proposal: Design an inhibitor that will inhibit iNOS from producing nitric oxide in hopes of reducing brain inflammation and the onset or progression of Parkinson's Disease. Parkinson’s Disease Protein of Interest 2: p3811 ● Mitogen-activated protein kinases (MAPK’s) are protein-serine/threonine kinases that are ubiquitous proline-directed. ● They play a role in signal transduction pathways that control intracellular processes. ● Such processes include hormonal and major developmental change as well as inflammation response. ● MAPK’s lie in protein kinase cascades. ● p38 is a type of MAPK that is used in the signalling transduction pathway.12 ● It plays a role in regulating cellular processes such as inflammation.13 Proposal: Design an inhibitor that will inhibit MAPK from being phosphorylated in hopes of reducing iNOS production, in turn reducing brain inflammation and the onset or progression of Parkinson's Disease. Biological Interest: Surfactant Protein Deficiency/Dysfunction ● A disorder of the lungs that causes breathing problems. ● The trouble breathing comes from alveoli collapse. ● Tissues around the alveoli of lungs stick together after exhalation if there is nonfunctional surfactant. ● Surfactant lowers the surface tension at the air-liquid interface in alveoli. 14 ● Surfactant forms a monolayer that disrupts the cohesiveness between liquid molecules, thus lowering surface tension. ● 15Interacts with and kills foreign particles and pathogens that enter the lungs. This protects the body from infections. ● Helps with the modulation of other immune responses. 16 ● Can have inheritance patterns depending on the specific genetic cause. ● SP-B, a type of surfactant deficiency, has the frequency of occurring in about 1 per 1 million infants. ● The main causes are mutations in several genes responsible for the production of surfactant. These genes are (SFTPC, ABCA3, and SFTPB). Protein of Interest: Human Surfactant protein ● Human Surfactant protein (SP-A, SP-B, SP-C, SP-D) Figure 4. Pulmonary Surfactant 17 Figure 5. Examples of Types of Surfactant Proteins18 ● Their special properties (“interfacially active proteins) are applicable to coatings on medical technologies (implants, nanodevices), and emulsifiers in other products. ● Surfactant proteins are usually divided into 3 groups ● 1. Functions through associated lipids - pulmonary surfactants ● 2. Small amphiphilic peptides ● 3. Non-lipid-associated globular proteins ● Another protein of interest is the surface-active proteins (fungal hydrophobins) in Schizophyllum commune. These proteins decrease surface tension at the air-water interface to mitigate this significant barrier to “hyphal erection”. This allows the hyphae to break the surface. ● Hydrophobins are smaller (7-9kDa) ● Low sequence similarity across hydrophobins ● 8 conserved Cysteine residues19 Proposal A. How do the lipids needed for pulmonary surfactants to function, associate with the protein? B. How does the structure of the various types of surfactant protein benefit their specific function? C. What do Cys residues forming 4 intramolecular disulphide bridges being conserved across all hydrophobins signify? Biological Interest: Thoracolumbar myelopathy in Pugs (PDM)20 ● Thoracolumbar myelopathy is a common chronic neurological issue in dogs which affect the spinal cord and vertebral column in pugs 20 ● Pugs often present with ataxia (without coordination), paraparesis (unable to move your legs), and incontinence (lack of voluntary control over urination or defecation)21 ● Thoracolumbar myelopathy occurs when a fluid-filled pocket develops on the outside of the spinal cord in the subarachnoid space, that becomes so dilated that it puts pressure on the spinal cord, which prevents messages from traveling normally, resulting in neurological signs22 ● Symptoms include: rear limb weakness and incoordination (ataxia), low tail carriage, incontinence, normal front limbs22 ● The pug-specific presentation of these symptoms (and phenotypes) suggests a genetic risk factor involved in Thoracolumbar myelopathy in pugs22 Protein of Interest: TRIM38 21 ● Tripartite motif protein 38, is a member of the TRIM family which is involved in key cellular processes such as proliferation, differentiation, apoptosis and more23 ● The encoded protein has a RING-type zinc finger, B box-type zinc finger and a SPRY domain23 ● Plays a crucial role in bone remodeling, and is involved in osteoblast metabolism21 (metabolic pathways such as glycolysis, Beta-oxidation, and amino acid metabolism24) ● TRIM38 regulates NF-kappa-B (involved in signaling pathways essential for normal cellular functions and development) activation through TAB2 degradation in osteoblasts ○ Osteoblasts are required for regulating skeletal development, maintenance, and bone remodeling21 Proposal: A. How to promote/regulate the activity of TRIM38 in Pugs with Thoracolumbar myelopathy B. How to positively induce NF-kappa-B Biological Interest: Botulism ● Rare neuroparalytic syndrome, potentially fatal ● Flaccid paralysis caused by exo-neurotoxin (BoNT) derived from the bacteria Clostridium botulinum (gram-positive, rod-shaped, spore-forming, obligate anaerobic bacterium) ● LD50 = 1-3 ng of toxin per kg of body mass ● Paralysis caused by irreversible inhibition of acetylcholine (ACh) release at the presynaptic nerve of the body's neuromuscular junctions (NMJs) ● Average of 162 annual cases between 2011-2015 in the US ● Infant Botulism caused by lack of robust immune system, allowing the growth of the C. botulinum in the digestive tract or bronchioles ○ BoNT migrates via transcytosis across the mucosal barrier (in either intestinal or pulmonary epithelium) into the circulation 25 ● Food-borne botulism caused by improperly stored foods, the bacteria remains in the intestinal tract (BoNT absorbed similarly to infantile botulism) ● Wound Botulism results from C. botulinum spore germination in devitalized tissue ● Once in bloodstream, BoNT migrates to and binds presynaptic nerve terminals of voluntary motor and autonomic neuromuscular joints 26 Protein of Interest: Botulinum Neurotoxin (BoNT) ● 150 kDa protein, comprised of a 100 kDa heavy chain and a 50 kDa light chain, linked by a single disulfide bridge ● Eight distinct serotypes (A-H), majority of the cases of botulism in the US are caused by serotypes A and B ● Heavy chain promotes endocytosis, Light chain is cleaved and enters the cytosol (usually of nerve cells) ● Light chain targets and cleaves serotype-specific target of the SNARE (SNAP-25, VAMP, or syntaxin) polypeptide complex (proteins required for fusion of ACh-containing vesicles with the presynaptic membrane) ○ Fusion allows exocytosis of ACh ○ Light chain considered a zinc-dependent endopeptidase 27 ● By cleaving these fusion complexes BoNT blocks presynaptic ACh release and inhibits muscle contractions 25 ● Elimination of BoNT can be enhanced by using serotype-specific neutralizing antibody (antitoxin), as it limits the number of SNARE complex affected by the toxin ● BoNT is sequestered by the liver and spleen ● Therapeutics against BoNT can target any of the three steps of mode of action of BoNT: binding, endocytosis/translocation, and endopeptidase activity 28 ○ First 2 steps carried out by Heavy Chain ○ Endopeptidase activity carried out by light chain 27 Proposal: A. How does the light chain locate serotype-specific sites on snare proteins and cleave them B. How does the heavy chain interact with other proteins to cause endocytosis a. how does the protein specifically target neurons Can botulinum be used as a favorable drug b. For what/how? C. Develop a molecular inhibitor against BoNT D. Re-engineer the heavy chain to direct molecules into nerve cells 25