Biology subjects

Svedruzic, Z. M.

Publications and source records attributed to Svedruzic, Z. M..

4 recordsLinked to original sources

bioRxiv2022

Binding of different substrate molecules at the docking site and the active site of γ-secretase can trigger toxic events in sporadic and familial Alzheimer's disease

Significancepathogenic changes in {gamma}-secretase activity, and its response to different drugs, can be greatly affected by changes in saturation of {gamma}-secretase with its substrate. The molecular mechanism is unclear. Resultsmultiscale molecular dynamics studies show that saturation of {gamma}-secretase with its substrate can result in parallel binding of different substrate molecules at the docking site and the active site. C-terminal domain of the second substrate can bind at cytosolic end of presenilin subunit while {gamma}-secretase is still processing its first substrate. Such interactions can disrupt dynamic presenilin structures that regulate proteolytic steps. Similar disruptions in dynamic presenilin structures can be produced by different drugs and by different disease-causing mutations. Thus, the presented two-substrate mechanism, can explain toxic inhibition of {gamma}-secretase activity and toxic increase in production of the longer, more hydrophobic, A{beta}-proteins. Toxic aggregation between N-terminal domains of the two substrates is controlled by nicastrin ectodomain. Such aggregation is more likely to happen with C99-{beta}CTF-APP than with C83-CTF-APP substrate, which can explain, why {beta}-secretase path is more pathogenic than -secretase path. The binding of C99-{beta}CTF-APP substrate to {gamma}-secretase can be controlled by substrate-channeling between nicastrin ectodomain and {beta}-secretase. ConclusionsThe presented two-substrate mechanism can explain why different studies consistently show that increase in saturation of {gamma}-secretase with its substrate can support pathogenic changes in different sporadic and familiar cases of the disease. Future drug-development strategies can target different physiological mechanisms that control the balance between cellular levels of {gamma}-secretase activity and the total amyloid metabolism.

neuroscience↗

bioRxiv2021

The Prion Protein Octarepeat Domain Forms Transient β-sheet Structures Upon Residue-Specific Cu(II) and Zn(II) Binding

Misfolding of the cellular prion protein (PrPC) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to play a crucial role in the protein misfolding, and metal imbalance may be part of TSE pathologies. PrPC is a combined Cu(II) and Zn(II) metal binding protein, where the main metal binding site is located in the octarepeat (OR) region. Here, we used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Upon metal binding, the OR region seems to adopt a transient antiparallel {beta}-sheet hairpin structure. Fluorescence spectroscopy data indicates that under neutral conditions, the OR region can bind both Cu(II) and Zn(II) ions, whereas under acidic conditions it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of both metal ions to the OR region results in formation of {beta}-hairpin structures. As formation of {beta}-sheet structures is a first step towards amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSEs.

biophysics↗

bioRxiv2020

Structural Analysis of Simultaneous Activation and Inhibition of Gamma-Secretase Activity in Development of Drugs for Alzheimers disease

SignificanceThe majority of drugs that target membrane-embedded protease {gamma}-secretase show unusual biphasic activation-inhibition dose-response in cells, model animals, and humans. Semagacestat and avagacestat are two biphasic-drugs that can facilitate cognitive decline in patients with Alzheimers disease. Initial mechanistic studies showed that the biphasic-drugs, and pathogenic mutations, can produce the same type of changes in {gamma}-secretase activity. ResultsDAPT, semagacestat LY-411,575, and avagacestat are four drugs that show different binding constants, and biphasic activation-inhibition dose-response curves, for amyloid-{beta}-40 products in SHSY-5 cells. Multiscale molecular dynamics studies showed that all four drugs bind to the most mobile parts in presenilin structure, at different ends of the 29 [A] long active site tunnel. Combined results from structure-activity studies, showed that the biphasic dose-response curves are a result of modulation of {gamma}-secretase activity by concurrent binding of multiple drug molecules at each end of the active site tunnel. The drugs activate {gamma}-secretase by forcing the active site tunnel to open, when the rate-limiting step is the tunnel opening, and formation of the enzyme-substrate complex. The drugs inhibit {gamma}-secretase as uncompetitive inhibitors, by binding next to the substrate to dynamic enzyme structures that regulate processive catalysis. The drugs can modulate the production of different amyloid-{beta} catalytic intermediates, by penetrating into the active site tunnel to different depth with different binding affinity. The drugs and pathogenic mutations affect the same dynamic processes in {gamma}-secretase structure. ConclusionsBiphasic-drugs like disease-causing mutations can reduce the catalytic capacity of {gamma}-secretase and facilitate pathogenic changes in amyloid metabolism.

neuroscience↗

bioRxiv2020

Substrate Channeling via a Transient Protein-Protein Complex: The case of D-Glyceraldehyde-3-Phosphate Dehydrogenase and L-Lactate Dehydrogenase

BackgroundD-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase (LDH) can form a complex that can regulate the major metabolic pathways, however, the exact mechanism remains unknown. We analyzed a possibility of NADH-channeling from GAPDH-NADH complex to LDH isozymes using enzymes from different cells. ResultsEnzyme-kinetics and NADH-binding studies showed that LDH can use GAPDH-NADH complex as a substrate. LDH activity with GAPDH-NADH complex was challenged with anti-LDH antibodies to show that the channeled and the diffusive reactions always take place in parallel. The channeling path is dominant only in assays with limiting free-NADH concertation that mimic cytosolic conditions. Analytical ultracentrifugation showed that the channeling does not require a high affinity complex. Molecular dynamics calculations and protein-protein interaction studies showed that LDH and GAPDH can form a leaky channeling complex only at subsaturating NADH concentrations. The interaction sites are conserved between LDH isozymes from heart and muscle, and between GAPDH molecules from rabbit and yeast cells. Positive electric fields between the NAD(H) binding sites on LDH and GAPDH tetramers, showed that NAD(H)-channeling within the LDH-GAPDH complex, can be an extension of NAD(H)-channeling between the adjacent subunits in each tetramer. ConclusionsIn the case of a transient (GAPDH-NADH)-LDH complex, the relative contribution from the channeled and the diffusive paths depends on the overlap between off-rates for the transient (GAPDH-NADH)-LDH complex and off-rates for the GAPDH-NADH complex. Molecular evolution or metabolic engineering protocols can exploit substrate channeling for metabolic flux control by fine-tuning substrate-binding affinity for the key enzymes in the competing reaction paths. Highlights- Substrate channeling molecular mechanism can regulate energy production and aerobic and anaerobic metabolism in cells - LDH and GAPDH can form a channeling complex only at sub-saturating NADH concentration - Channeled and diffusive paths always compete and take place in parallel - NADH channeling does not require a high-affinity complex - NADH channeling within GAPDH-LDH complex is an extension of NAD(H) channeling within each tetramer - Allosteric modulation of NADH binding affinity in GAPDH tetramer can regulate NAD(H) channeling

biochemistry↗