Biology subjects

Devkota, S.

Publications and source records attributed to Devkota, S..

16 recordsLinked to original sources

bioRxiv2026

Tryptophan degradation by intestinal Bacteroides induces anti-tumor immunity and limits melanoma growth

Defining mechanisms used by gut microbiota to control anti-tumor immunity may offer novel therapeutic modalities. Here, we demonstrate that Bacteroides rodentium and closely related Bacteroides uniformis species induce anti-tumor immunity and limit melanoma development when colonized in either germ-free (GF) mice, mice with a complex microbiome, or WT mice. Enhanced CD8+ T cell infiltration seen in tumors of mice harboring B. rodentium coincided with increased expression of immune-stimulating pathways and activation of bone marrow-derived dendritic cells that were co-cultured with the B. rodentium secretome. Metabolomic analyses of cecal samples from GF mice colonized with Altered Shedlar Flora (ASF) plus B. rodentium revealed lower tryptophan levels than in ASF-colonized controls, and WT mice fed a tryptophan-deficient diet exhibited inhibition of melanoma development. In silico genomic reconstruction of metabolic pathways revealed that both B. rodentium and B. uniformis harbor tryptophanase A (TnaA) and aromatic amino transferase (ArAT) genes, both of which function in tryptophan degradation. Administration of a B. uniformis harboring TnaA mutant failed to inhibit melanoma growth in gnotobiotic mice. Notably, administration of indoles, but not kynurenines, also effectively inhibited melanoma development, increasing immune cell infiltration into the tumors. Correspondingly, levels of bacterially encoded tryptophan-degrading enzymes were higher in cohorts of melanoma patients responding to immune checkpoint blockade. These findings highlight a novel mechanism of anti-tumor immunity and tumor growth inhibition dependent on the tryptophan degradation products, indoles, produced by intestinal Bacteroides species.

cancer biology↗

bioRxiv2026

Bridge recombinase enables versatile rewriting of bacterial genomes

Bacteria drive crucial processes across ecosystems and impact human health, yet tools to rewrite microbiomes remain limited. Here, we show that bridge recombinase enables versatile and programmable genome editing across the bacterial tree of life. In Escherichia coli, we achieved 142 kb insertions at >90% efficiency, megabase-scale inversions (2.3 Mb), and pathway-scale 50 kb excisions. With a single ortholog and bridge RNA (bRNA), we edited bacterial isolates spanning five phyla and diverse members of two human gut communities. We overcame cross-reactivity between co-expressed bRNAs to establish search-and-replace Targetable Recombinase Assisted DNA Exchange (TRADE) editing and demonstrated capture and interphylum transfer of chromosomal pathways, enabling programmable horizontal gene transfer. These advances establish bridge recombinase as a foundation for reprogramming gene flow in complex microbial communities.

synthetic biology↗

bioRxiv2026

Pan-repository analysis reveals a drug-activating function of microbial bile acid conjugation

Microbially modified bile acids shape host physiology by regulating nutrient absorption, glucose homeostasis, circadian rhythms and thermoregulation. Here we identify a previously unrecognized drug-activating function of microbial bile acid conjugation. By systematically mining human LC-MS/MS datasets across public repositories and linking uncharacterized bile acid spectra to health-associated metadata, we discovered conjugates of the >75-year-old anti-inflammatory drug 5-aminosalicylic acid (5-ASA) with primary and secondary bile acids, including cholic, deoxycholic and lithocholic acids. These bile acid-drug conjugates were detected specifically in individuals treated with 5-ASA or its prodrugs. Multiple gut bacteria, including members of the Bacteroidota and Bacillota, generated cholyl-5-ASA in vitro, and bile salt hydrolase-associated transaminase activity was required for conjugate formation. In a mouse model of colitis, cholyl-5-ASA was associated with reduced intestinal inflammatory pathology and showed markedly enhanced activation of PPAR-{gamma} in cell-based reporter assays compared with 5-ASA alone. Consistent with this activity, cholyl-5-ASA elicited selective immunophenotypic changes in CD4 T cells in vitro, including increased Foxp3+ regulatory T cells. Together with prior evidence that 5-ASA efficacy depends on the microbiome, these findings support a model in which microbial bile acid conjugation represents a key activation step for 5-ASA therapy. More broadly, this work demonstrates how pan-repository metabolomics can uncover previously unrecognized microbiome-dependent chemical functions with direct therapeutic relevance.

biochemistry↗

bioRxiv2024

Alzheimer-mutant γ-secretase complexes stall amyloid β-peptide production

Missense mutations in the amyloid precursor protein (APP) and presenilin-1 (PSEN1) cause early-onset familial Alzheimers disease (FAD) and alter proteolytic production of secreted 38-to-43-residue amyloid {beta}-peptides (A{beta}) by the PSEN1-containing {gamma}-secretase complex, ostensibly supporting the amyloid hypothesis of pathogenesis. However, proteolysis of APP substrate by {gamma}-secretase is processive, involving initial endoproteolysis to produce long A{beta} peptides of 48 or 49 residues followed by carboxypeptidase trimming in mostly tripeptide increments. We recently reported evidence that FAD mutations in APP and PSEN1 cause deficiencies in early steps in processive proteolysis of APP substrate C99 and that this results from stalled {gamma}-secretase enzyme-substrate and/or enzyme-intermediate complexes. These stalled complexes triggered synaptic degeneration in a C. elegans model of FAD independently of A{beta} production. Here we conducted full quantitative analysis of all proteolytic events on APP substrate by {gamma}-secretase with six additional PSEN1 FAD mutations and found that all six are deficient in multiple processing steps. However, only one of these (F386S) was deficient in certain trimming steps but not in endoproteolysis. Fluorescence lifetime imaging microscopy in intact cells revealed that all six PSEN1 FAD mutations lead to stalled {gamma}-secretase enzyme-substrate/intermediate complexes. The F386S mutation, however, does so only in A{beta}-rich regions of the cells, not in C99-rich regions, consistent with the deficiencies of this mutant enzyme only in trimming of A{beta} intermediates. These findings provide further evidence that FAD mutations lead to stalled and stabilized {gamma}-secretase enzyme-substrate and/or enzyme-intermediate complexes and are consistent with the stalled process rather than the products of {gamma}-secretase proteolysis as the pathogenic trigger.

biochemistry↗

bioRxiv2024

The intestinal microbiota contributes to the development of immune-mediated cardiovascular inflammation and vasculitis in mice.

Alterations in the intestinal microbiota contribute to the pathogenesis of various cardiovascular disorders, but how they affect the development of Kawasaki disease (KD), an acute pediatric vasculitis, remains unclear. We report that depleting the gut microbiota reduces the development of cardiovascular inflammation in a murine model mimicking KD vasculitis. The development of cardiovascular lesions was associated with alterations in the intestinal microbiota composition and, notably, a decreased abundance of Akkermansia muciniphila and Faecalibacterium prausnitzii. Oral supplementation with either of these live or pasteurized individual bacteria, or with short-chain fatty acids (SCFAs) produced by them, attenuated cardiovascular inflammation. Treatment with Amuc_1100, the TLR-2 signaling outer membrane protein from A. muciniphila, also decreased the severity of vascular inflammation. This study reveals an underappreciated gut microbiota-cardiovascular inflammation axis in KD vasculitis pathogenesis and identifies specific intestinal commensals that regulate vasculitis in mice by producing metabolites or via extracellular proteins acting on gut barrier function. IN BRIEFIt remains unclear whether changes in the intestinal microbiota composition are involved in the development of cardiovascular lesions associated with Kawasaki disease (KD), an immune-mediated vasculitis. Jena et al. observe alterations in the intestinal microbiota composition of mice developing vasculitis, characterized by reduced A. muciniphila and F. prausnitzii. Oral supplementation with either of these bacteria, live or pasteurized, or with bacteria-produced short-chain fatty acids (SCFAs) or Amuc_1100, the TLR-2 signaling outer membrane protein of A. muciniphila, was sufficient to alleviate the development of cardiovascular lesions in mice by promoting intestinal barrier function. HIGHLIGHTSO_LIAbsence or depletion of the microbiota decreases the severity of vasculitis in a murine model mimicking KD vasculitis. C_LIO_LISupplementation of B. wadsworthia and B. fragilis promotes murine KD vasculitis. C_LIO_LIDecreased abundances of F. prausnitzii and A. muciniphila are associated with the development of cardiovascular lesions in mice. C_LIO_LISupplementation with either live or pasteurized A. muciniphila and F. prausnitzii, or the TLR-2 signaling Amuc_1100, reduces the severity of vasculitis by promoting gut barrier function. C_LI

immunology↗

bioRxiv2023

Microbial aromatic amino acid metabolism is modifiable in fermented food matrices to promote bioactivity

Ingestion of fermented foods impacts human immune function, yet the bioactive food components underlying these effects are not understood. Here, we interrogated whether fermented food bioactivity could be traced to a class of microbial metabolites derived from aromatic amino acids (ArAA), termed aryl-lactates. Using targeted metabolomics, we established that the aryl-lactates phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (4-HPLA), and indole-3-lactic acid (ILA), are present in varying concentrations across a wide range of commercially available fermented foods, including many vegetable and dairy ferments. After pinpointing fermented food-associated lactic acid bacteria (LAB) that produce high levels of aryl-lactates (e.g., Lactiplantibacillus plantarum), we utilized our knowledge of LAB metabolism to identify fermentation conditions (added cultures [e.g., L. plantarum] and metabolic co-factors [e.g., aryl-pyruvates]) to increase aryl-lactate production in food matrices up to 5x103 fold vs. standard fermentation conditions. Next, using ex vivo reporter assays, we found that a variety of food matrix conditions optimized for aryl-lactate production exhibited enhanced agonist activity for the human aryl-hydrocarbon receptor (AhR) as compared to standard fermentation conditions and/or commercial brands. Moreover, we determined that strategies to enhance aryl-lactates effectively maintained food matrix AhR bioactivity across 4 weeks of storage. Reduced microbial-induced AhR activity has emerged as a hallmark of many chronic inflammatory diseases, thus we envision strategies to enhance microbially produced aryl-lactates and thus AhR bioactivity of fermented foods can be leveraged to improve human health.

microbiology↗

bioRxiv2023

Microplastics are detected in bull and dog sperm and polystyrene microparticles impair sperm fertilization

The alarming increase in global infertility rates has coincided with the pervasive accumulation of microplastics (MPs) resulting from the poor management of plastic waste. This concerning trend is particularly troubling because only 10% of male infertility cases can be attributed to identifiable causes, leaving a significant knowledge gap in our understanding of their underlying factors. To bridge this critical gap, it is important to explore the connection between the accumulation of MPs and the observed decline in male fertility. Here, the presence of microplastics in reproductive fluids from bulls and dogs was assessed and used as baseline concentrations for bull sperm exposure. Bovine epididymal sperm (ES) presented a mean of 72.5 MP particles mL-1 (0.3691 g mL-1) while canine seminal plasma had an average of 35.4 MP particles mL-1 (0.0066 g mL-1). Bovine sperm was exposed to three different concentrations of a mixture of 1.1, 0.5, and 0.3 {micro}m polystyrene (PS) beads: (1) 0.7 g mL-1, blood concentration of PS in cows (bPS); (2) 0.37 g mL-1, concentration of total MPs in ES (esMP); and (3) 0.026 g mL-1, concentration of PS in ES (esPS). All sperm samples incubated with PS exhibited reduced motility compared with the CT at 0.5 h. However, PS exposure did not affect acrosome or induced oxidative stress. When used for in vitro fertilization, the sperm exposed to PS had decreased blastocyst rates, in addition to inducing ROS formation and apoptosis on resulting embryos. By employing realistic exposure concentrations, this research sought to shed light on the comprehensive impact of MPs on bovine sperm and the quality of resulting embryos, providing the first evidence of MPs in bovine and dog sperm and demonstrating the detrimental effect of PS MPs on sperm motility and functionality. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/571802v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@5871acorg.highwire.dtl.DTLVardef@8ee9f2org.highwire.dtl.DTLVardef@a1416borg.highwire.dtl.DTLVardef@cde261_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMicroplastics were found across the most diverse range of environments and their presence have been shown to affect reproductive parameters within different species. C_LIO_LITrue-to-life concentrations of exposure were used to assess the potential effects of polystyrene in sperm parameters and fertilization. C_LIO_LIPolystyrene microplastics attach to sperm and decrease motility, also reducing sperm functionality as seen by decreased blastocyst rate and increased oxidative stress in embryos. C_LI

developmental biology↗

bioRxiv2023

Mechanical Properties of Native and Decellularized Reproductive Tissues: Insights for Tissue Engineering Strategies

Understanding the mechanical properties and porosity of reproductive tissues is vital for regenerative medicine in tissue engineering. This study investigated the changes in Youngs modulus (YM), storage modulus (E'), loss modulus (E''), and porosity of native and decellularized bovine reproductive tissues during the estrous cycle. Testis tunica albuginea had significantly higher YM, E', and E'' than the inner testis, indicating greater stiffness and viscoelasticity. Endometrium showed no distinct differences in YM, E', or E' across the estrous cycle or between horns. Ovaries exhibited significant variations in YM, E', E'', and porosity, with higher YM and E' in the ipsilateral cortex and medulla during the luteal phase. Decellularized ovarian tissues displayed increased porosity. The oviduct displayed no significant differences in YM or E' in the isthmus, but the contralateral ampulla had reduced YM and E' in the luteal phase. These findings offer valuable insights into the dynamic mechanical properties and porosity of reproductive tissues, facilitating the development of biomimetic scaffolds for tissue engineering applications.

bioengineering↗

bioRxiv2023

Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes

Alzheimers disease is characterized pathologically by cerebral deposition of 42-residue amyloid {beta}-peptide (A{beta}42), proteolytically produced from amyloid precursor protein (APP) by {beta}- and {gamma}-secretases.1 Although mutations in APP and presenilin, the catalytic component of {gamma}-secretase, cause familial Alzheimers disease (FAD), a role for A{beta}42 as the primary disease driver has not been clearly established and remains controversial.2,3 Here we show through comprehensive analysis of the multi-step proteolysis of APP substrate C99 by {gamma}-secretase that FAD mutations are consistently deficient in early proteolytic events, not later events that produce secreted A{beta} peptides. Cryo-electron microscopy revealed that a substrate mimetic traps {gamma}-secretase at the transition state for intramembrane proteolysis, and this structure closely aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and fluorescence lifetime imaging microscopy in cultured cells support stabilization by FAD mutations of enzyme-substrate and/or enzyme-intermediate complexes. Neuronal expression of C99 and/or presenilin-1 in Caenorabditis elegans led to age-dependent synaptic loss only when one of the transgenes carried an FAD mutation. Designed mutations that stabilize the enzyme-substrate complex and block proteolysis likewise led to synaptic loss. Collectively, these findings implicate the stalled process--not the released products--of {gamma}-secretase cleavage of substrates in FAD pathogenesis.

neuroscience↗

bioRxiv2022

The molecular consequences of androgen activity in the human breast

The mammary gland has been extensively studied for estrogen and progesterone reactivity, but the molecular effects of androgen in the breast remain largely unexplored. Transgender men are recorded female at birth but identify as male and may undergo gender-affirming androgen therapy to align their physical characteristics and gender identity. Here we perform single cell resolution transcriptome, chromatin, and spatial profiling of androgen treated breasts from transgender men. We find male-biased androgen receptor gene targets are upregulated in cells expressing androgen receptor, and that paracrine signaling drives sex-relevant changes in other cell types. We observe an altered epithelium, shifts in immune populations, and a reduction of capillary vasculature. Finally, we find evidence of the metabolic impact of androgen and identify a gene regulatory network driving androgen-directed fat loss. This work elucidates the molecular consequences of androgen in the human breast at single cell resolution.

genomics↗

bioRxiv2021

Systemic IgG repertoire as a biomarker for translocating gut microbiota members

While the microbiota has been associated with diseases states, how specific alterations in composition, function, or localization contribute to pathologies remains unclear. The ability of defined microbes to translocate has been linked to diseases including inflammatory bowel disease (IBD) and was shown to promote responses to immune checkpoint therapy. However, scalable and unbiased tools to uncover microbes with enhanced ability to translocate are limited. Herein, we developed an approach to utilize systemic IgG in an unbiased, culture-independent, and high-throughput fashion as a biomarker to identify gut microbiota members that are capable of translocation across the gastrointestinal barrier. We validate these findings in a cohort of human subjects, and highlight a number of microbial taxa against which elevated IgG responses are unique to subjects with IBD including Collinsella, Bifidobacterium, Faecalibacterium, and Blautia spp. Collinsella and Bifidobacterium taxa identified as translocators and targets of immunity in IBD also exhibited heightened bacterial activity and growth rates in Crohns disease subjects. Our approach may represent a complementary tool to illuminate privileged interactions between host and its microbiota, and may provide an additional lens by which to uncover microbes linked to disease processes. One Sentence SummaryCirculating microbiota-specific IgG can identify gut microbiota constituents capable of crossing the gut barrier.

immunology↗

bioRxiv2021

Mechanism of Tripeptide Trimming by γ-Secretase

The membrane-embedded {gamma}-secretase complex processively cleaves within the transmembrane domain of amyloid precursor protein (APP) to produce 37-to-43-residue amyloid {beta}-peptides (A{beta}) of Alzheimers disease (AD). Despite its importance in pathogenesis, the mechanism of processive proteolysis by {gamma}-secretase remains poorly understood. Here, mass spectrometry and western blotting were used to quantify the efficiency of the first tripeptide trimming step (A{beta}49[->]A{beta}46) of wildtype (WT) and familial AD (FAD) mutant APP substrate. In comparison to WT APP, the efficiency of this first trimming step was similar for the I45F, A42T and V46F APP FAD mutants, but substantially diminished for the I45T and T48P mutants. In parallel with biochemical experiments, all-atom simulations using a novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method were applied to investigate tripeptide trimming of A{beta}49 by {gamma}-secretase. The starting structure was active {gamma}-secretase bound to A{beta}49 and APP intracellular domain (AICD), as generated from our previous study that captured activation of {gamma}-secretase for the initial endoproteolytic cleavage of APP (Bhattarai et al., ACS Cent Sci, 2020, 6:969-983). Pep-GaMD simulations captured remarkable structural rearrangements of both the enzyme and substrate, in which hydrogen-bonded catalytic aspartates and water became poised for tripeptide trimming of A{beta}49 to A{beta}46. These structural changes required a positively charged N-terminus of endoproteolytic coproduct AICD, which could dissociate during conformational rearrangements of the protease and A{beta}49. The simulation findings were highly consistent with biochemical experimental data. Taken together, our complementary biochemical experiments and Pep-GaMD simulations have enabled elucidation of the mechanism of tripeptide trimming by {gamma}-secretase. Significance statementProduction of amyloid {beta}-peptide (A{beta}) of Alzheimers disease (AD) from its precursor protein requires a series of proteolytic events carried out by the membrane-embedded {gamma}-secretase complex. Mutations in the substrate and enzyme that produce A{beta} cause hereditary AD and these mutations affect tripeptide trimming of initially formed long A{beta} peptides by {gamma}-secretase. Little is known about the structural mechanism of this trimming process. Here, we have developed a molecular dynamic model for the first step of trimming (A{beta}49 to A{beta}46) by {gamma}-secretase. Conformational changes in the enzyme-substrate complex to set up this trimming step require the presence of N-terminally charged endoproteolytic cleavage co-product. Computational effects of AD-causing mutations in A{beta}49 on the trimming process were validated through biochemical experiments.

biochemistry↗

bioRxiv2021

Design of Transmembrane Mimetic Structural Probes to Trap Different Stages of γ-Secretase-Substrate Interaction

The transmembrane domain (TMD) of the amyloid precursor protein of Alzheimers disease is processively cut by {gamma}-secretase through endoproteolysis and tricarboxypeptidase "trimming". We recently developed a prototype substrate TMD mimetic for structural analysis--composed of a helical peptide inhibitor linked to a transition-state analog--that simultaneously engages a substrate exosite and the active site and is pre-organized to trap the carboxypeptidase transition state. Here we developed variants of this prototype designed to allow visualization of transition states for endoproteolysis, TMD helix unwinding, and lateral gating of substrate, identifying potent inhibitors for each class. These TMD mimetics exhibited non-competitive inhibition and occupy both exosite and active site as demonstrated by inhibitor cross competition experiments and photoaffinity probe binding assays. The new probes should be important structural tools for trapping different stages of substrate recognition and processing via ongoing cryo-electron microscopy with {gamma}-secretase, ultimately aiding rational drug design.

biochemistry↗

bioRxiv2021

Seasonal Abundance and Spatial Pattern of Distribution of Liriomyza trifolii (Diptera: Agromyzidae) and Its Parasitoid on Bean and Squash in South Florida.

American serpentine leafminer, Liriomyza trifolii, is a polyphagous insect pest that feeds on a wide range of vegetable and ornamental plants around the world. To develop an effective IPM program, information on the seasonal field distribution and population dynamics of leafminer and its parasitoids is very important. Therefore, seasonal abundances and spatial distributions of, L. trifolii on snap bean and squash were studied during four crop growing periods between 2013 to 2015 in Homestead, Florida. The mean numbers of mines, larvae, pupae, emerged adults, and parasitoids on snap bean were highest at 2 weeks after planting during all four growing periods. Whereas, the mean numbers of mines, larvae, pupae, emerged adults, and parasitoids on squash were highest at 3 weeks after planting during all four growing periods. L. trifolii distributions tended to be aggregated (1 < b/{beta}) on snap bean at 2 weeks after planting during all four growing periods but had uniform (1 > b/{beta}) distributions on squash at 2 weeks after planting during all four growing periods. Similar results were seen on the distribution of leafminer parasitoids on both bean and squash.

plant biology↗

bioRxiv2020

Development of early-stage type 1 diabetes in germ-free interleukin-10 deficient mice

Several experimental models demonstrate a role for gut microbiota in the progression of type 1 diabetes (T1D) in genetically prone hosts. While the association between disturbances in gut microbiota, or microbial dysbiosis, and complex immune diseases such as inflammatory bowel diseases (IBD) are well established, less is known about its role in T1D pathogenesis. In IBD-prone interleukin-10 deficient (IL-10 KO) mice, the absence of gut microbiota under germ-free (GF) conditions prevents IBD development. However, in aged GF IL-10 KO mice (>6-months of age), polyuria and pancreatic lymphocytic infiltration resembling T1D lesions was observed. Approximately 50% of male and female mice above 6-months of age develop pancreatic immune cell infiltration, as compared to none in conventionally-raised and fecal microbiota transplanted (FMT) IL-10 KO counterparts. Immunofluorescence staining of islet infiltrates was positive for adaptive and innate immunological markers, including lymphoid and myeloid cell markers, which typically characterize autoimmune T1D lesions. A subset of GF IL-10 KO mice was also positive for insulin autoantibodies (IAA), but the majority of mice did not become diabetic. Our findings of early stage lymphocytic infiltrates in the pancreas and IAA in the absence of overt diabetes in GF IL-10 KO mice embody the early stages of T1D pathogenesis. As such, we propose that the presence of gut microbiota play a protective role against immune infiltration in the pancreas of genetically prone hosts. Moreover, our model provides an opportunity to better understand the role of the microbiota in the early stages of immune pathogenesis and perhaps conceive the development of microbe-mediated prophylactic strategies to treat or even prevent T1D.

physiology↗

bioRxiv2020

Mechanisms of γ-Secretase Activation and Substrate Processing

Amyloid {beta}-peptide, the principal component of characteristic cerebral plaques of Alzheimers disease (AD), is produced through intramembrane proteolysis of the amyloid precursor protein (APP) by {gamma}-secretase. Despite the importance in pathogenesis of AD, the mechanisms of intramembrane proteolysis and substrate processing by {gamma}-secretase remain poorly understood. Here, complementary all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method and biochemical experiments were combined to investigate substrate processing of wildtype and mutant APP by {gamma}-secretase. The GaMD simulations captured spontaneous activation of {gamma}-secretase, with hydrogen bonded catalytic aspartates and water poised for proteolysis of APP at the {varepsilon} cleavage site. Furthermore, GaMD simulations revealed that familial AD mutations I45F and T48P enhanced the initial {varepsilon} cleavage between residues Leu49-Val50, while M51F mutation shifted the {varepsilon} cleavage site to the amide bond between Thr48-Leu49. Detailed analysis of the GaMD simulations allowed us to identify distinct low-energy conformational states of {gamma}-secretase, different secondary structures of the wildtype and mutant APP substrate, and important active-site sub-pockets for catalytic function of the enzyme. The simulation findings were highly consistent with experimental analyses of APP proteolytic products using mass spectrometry and western blotting. Taken together, the GaMD simulations and biochemical experiments have enabled us to elucidate the mechanisms of {gamma}-secretase activation and substrate processing, which should facilitate rational computer-aided drug design targeting this functionally important enzyme.

biochemistry↗