Biology subjects

Nguyen, H.

Publications and source records attributed to Nguyen, H..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Cryo-EM structures of human SID-1 transmembrane family proteins and implications for their low-pH-dependent RNA transport activity

Human SIDT1 and SIDT2 are closely related members of the systemic RNA interference (RNAi)-defective (SID-1) transmembrane family. Both mediate RNA internalization and intracellular transport and are involved in various biological processes. However, the molecular basis of RNA uptake, especially for exogenous small RNAs, remains elusive. Here, we present the cryo-electron microscopy (cryo-EM) structures of human SIDT1 and SIDT2. Both structures reveal the overall architecture of a dimeric arrangement contributed by the {beta}-strand-rich extracellular domain (ECD) and the transmembrane domain (TMD) with 11 passes, highlighting the remarkable structural congruence. The in situ assays confirm that SIDT1 and SIDT2 exist as dimers or higher-order oligomers. We demonstrate that for both SIDT1 and SIDT2, the ECD binds small RNAs, such as dietary plant-derived miRNA, only under acidic conditions. In addition, RNA binding under low pH can trigger higher-order assembly of the ECD dimer, suggesting the potential importance of oligomerization during RNA uptake. Our results illustrate the molecular features of the conserved SID-1 family proteins to elucidate the mechanism of the low pH-dependent activation of RNA uptake mediated by SIDT1 and SIDT2. This study provides a promising understanding of the molecular basis of the nucleic acid delivery platform, which may potentially open new avenues for the design and optimization of RNA-based therapies.

biochemistry↗

bioRxiv2023

Thiazolidinediones are partially effective bitter blockers

PurposeThe bad bitter taste of some medicines is a barrier to overcoming non-compliance with medication use, especially life-saving drugs given to children and the elderly. Here we evaluated a new class of bitter blockers (thiazolidinediones; TZDs). MethodsIn this study, two TZDs were tested, rosiglitazone (ROSI) and a simpler form of TZD, using a high-potency sweetener as a positive control (neohesperidin dihydrochalcone, NHDC). We tested bitter-blocking effects using the bitter drugs tenofovir alafenamide fumarate (TAF), a treatment for HIV and hepatitis B infection, and praziquantel (PRAZ), a treatment for schistosomiasis, by conducting taste testing with two separate taste panels: a general panel (N=97, 20-23 yrs, 82.5% female, all Eastern European) and a genetically informative panel (N=158, including 68 twin pairs, 18-82 yrs, 76% female, 87% European ancestry). Participants rated the bitterness intensity of the solutions on a 100-point generalized visual analog scale. FindingsParticipants in both taste panels rated the bitter drugs TAF and PRAZ as less bitter on average when mixed with NHDC than when sampled alone. ROSI partially suppressed the bitterness of TAF and PRAZ, but effectiveness differed between the two panels: bitterness was significantly reduced for PRAZ but not TAF in the general panel and for TAF but not PRAZ in the genetically informative panel. ROSI was a more effective blocker than the other TZD. ImplicationsThese results suggest that TZDs are partially effective bitter blockers, suggesting other TZDs should be designed and tested with more drugs and on diverse populations to define which ones work best with which drugs and for whom. The discovery of bitter receptor blockers can improve compliance with medication use.

physiology↗

bioRxiv2023

Rapid and automated design of two-component protein nanomaterials using ProteinMPNN

The design of novel protein-protein interfaces using physics-based design methods such as Rosetta requires substantial computational resources and manual refinement by expert structural biologists. A new generation of deep learning methods promises to simplify protein-protein interface design and enable its application to a wide variety of problems by researchers from various scientific disciplines. Here we test the ability of a deep learning method for protein sequence design, ProteinMPNN, to design two-component tetrahedral protein nanomaterials and benchmark its performance against Rosetta. ProteinMPNN had a similar success rate to Rosetta, yielding 13 new experimentally confirmed assemblies, but required orders of magnitude less computation and no manual refinement. The interfaces designed by ProteinMPNN were substantially more polar than those designed by Rosetta, which facilitated in vitro assembly of the designed nanomaterials from independently purified components. Crystal structures of several of the assemblies confirmed the accuracy of the design method at high resolution. Our results showcase the potential of deep learning-based methods to unlock the widespread application of designed protein-protein interfaces and self-assembling protein nanomaterials in biotechnology.

biochemistry↗

bioRxiv2023

Comprehensive analysis of network reconstruction approaches based on correlation in metagenomic data

Microbiome analysis is transforming our understanding of biological processes related to human health, epidemiology (antimicrobial resistance, horizontal gene transfer) environmental and agricultural studies. At the core of microbiome analysis is the description of microbial communities based on quantification of microbial taxa and dynamics. In the study of bacterial abundances, it is becoming more relevant to consider their relationship, to embed these data in the framework of network theory, allowing characterization of features like node relevance, pathway and community structure. In this work we characterize the principal biases in reconstructing networks from correlation measures, associated with the compositional character of relative abundance data, the diversity of abundances and the presence of unobserved species within a single sample, that might lead to wrong correlation estimates. We show how most of these problems can be overcome by applying typical transformations for compositional data, that allow the application of simple measures such as Pearsons correlation to correctly identify the positive and negative relationships between relative abundances, when data dimensionality is sufficiently high. Some issues remain, like the role of data sparsity, that if not properly addressed can lead to imbalances in correlation coefficient distribution.

systems biology↗

bioRxiv2023

Hepatic lipid-associated macrophages mediate the protective effects of bariatric surgery against NASH

For patients with obesity and metabolic syndrome, bariatric procedures such as vertical sleeve gastrectomy (VSG) have a clear benefit in ameliorating metabolic dysfunction-associated steatohepatitis (MASH). While the effects of bariatric surgeries have been mainly attributed to nutrient restriction and malabsorption, whether immuno-modulatory mechanisms are involved remains unclear. Here we report that VSG ameliorates MASH progression in a weight loss- independent manner. Single-cell RNA sequencing revealed that hepatic lipid-associated macrophages (LAMs) expressing the triggering receptor expressed on myeloid cells 2 (TREM2) increase their lysosomal activity and repress inflammation in response to VSG. Remarkably, TREM2 deficiency in mice ablates the reparative effects of VSG, suggesting that TREM2 is required for MASH resolution. Mechanistically, TREM2 prevents the inflammatory activation of macrophages and is required for their efferocytotic function. Overall, our findings indicate that bariatric surgery improves MASH through a reparative process driven by hepatic LAMs, providing insights into the mechanisms of disease reversal that may result in new therapies and improved surgical interventions.

physiology↗

bioRxiv2023

5-ASA can functionally replace Clostridia to prevent a post-antibiotic bloom of Candida albicans by maintaining epithelial hypoxia

Antibiotic prophylaxis sets the stage for an intestinal bloom of Candida albicans, which can progress to invasive candidiasis in patients with hematologic malignancies. Commensal bacteria can reestablish microbiota-mediated colonization resistance after completion of antibiotic therapy, but they cannot engraft during antibiotic prophylaxis. Here we use a mouse model to provide a proof of concept for an alternative approach, which replaces commensal bacteria functionally with drugs to restore colonization resistance against C. albicans. Streptomycin treatment, which depletes Clostridia from the gut microbiota, disrupted colonization resistance against C. albicans and increased epithelial oxygenation in the large intestine. Inoculating mice with a defined community of commensal Clostridia species reestablished colonization resistance and restored epithelial hypoxia. Notably, these functions of commensal Clostridia species could be replaced functionally with the drug 5-aminosalicylic acid (5-ASA), which activates mitochondrial oxygen consumption in the epithelium of the large intestine. When streptomycin-treated mice received 5-ASA, the drug reestablished colonization resistance against C. albicans and restored physiological hypoxia in the epithelium of the large intestine. We conclude that 5-ASA treatment is a non-biotic intervention that restores colonization resistance against C. albicans without requiring the administration of live bacteria.

microbiology↗

bioRxiv2023

Binding of SARS-CoV-2 non-structured protein 1 to 40S ribosome inhibits mRNA translation

Experiments have shown that non-structural protein 1 (NSP1) of SARS-CoV-2 is a factor that restricts cellular gene expression and prevents mRNA translation in the ribosome 40S subunit. However, the molecular mechanism of this phenomenon remains unclear. To clarify this issue, all-atom steered molecular dynamics and coarse-grained alchemical simulations were used to compare the binding affinity of mRNA to 40S ribosome in the absence and presence of NSP1. We found that NSP1 binding to the 40S ribosome dramatically increases the binding affinity of mRNA, which, in agreement with experiment, suggests that NSP1 can stall mRNA translation. The mRNA translation has been found to be driven by electrostatic mRNA-40S ribosome interactions. Water molecules have been demonstrated to play an important role in stabilizing the mRNA-40S ribosome complex. The NSP1 residues that are critical in triggering a translation arrest have been identified.

biophysics↗

bioRxiv2023

Design of stimulus-responsive two-state hinge proteins

Proteins that switch between two structural states as a function of environmental stimuli are widespread in nature. These proteins structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Engineering challenges ranging from biological computing devices to molecular motors require such two-state switches, but designing these is an unsolved problem as it requires sculpting an energy landscape with two low-energy but structurally distinct conformations that can be modulated by external inputs. Here we describe a general design approach for creating "hinge" proteins that populate one distinct state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, and double electron-electron resonance spectroscopy demonstrate that despite the significant structural differences, the two states are designed with atomic level accuracy. The kinetics and thermodynamics of effector binding can be finely tuned by modulating the free energy differences between the two states; when this difference becomes sufficiently small, we obtain bistable proteins that populate both states in the absence of effector, but collapse to a single state upon effector addition. Like the transistor, these switches now enable the design of a wide array of molecular information processing systems.

biophysics↗

bioRxiv2023

Characterizing the microbiome of patients with myeloproliferative neoplasms during a Mediterranean diet intervention

Myeloproliferative neoplasms (MPN) are a class of hematological malignancies which result in the overproduction of myeloid lineage cells. These malignancies result in increased cytokine production and inflammation, which correlate with worsened symptom burden and prognosis. Other than bone marrow transplantation, there is no cure for myeloproliferative neoplasms. As such, treatments focus on reducing thrombotic risk, inflammation, and symptom burden. Because current pharmacological treatments carry significant side-effects, there is a need to explore low-risk therapies. One alternative is the Mediterranean diet, which is rich in anti-inflammatory foods, reduces inflammatory biomarkers, and beneficially alters the gut microbiome. Here, we performed a 15-week clinical trial of 28 individuals with MPN who were randomized to dietary counseling based on either a Mediterranean diet or standard U.S. Guidelines for Americans. Our primary objective was to determine if MPN patients were able to adopt a Mediterranean eating style when supported with dietician counseling. As exploratory endpoints, we investigated the impact of diet and inflammation on the gut microbiome. Using shotgun metagenomic sequencing, we found that microbiome diversity and composition were stable throughout the study duration in both cohorts. Furthermore, we discovered significant differences in the microbiomes between MPN subtypes, such as increased beta-dispersion in subjects with myelofibrosis. Lastly, we found several significant correlations between the abundances of multiple bacterial taxa and cytokine levels. Together, this study provides insight into the interaction between diet, inflammation, and the gut microbiome. ImportanceThe gut microbiome serves as an interface between the host and diet. Diet and the gut microbiome both play important roles in managing inflammation, which is a key aspect of MPN. Studies have shown that a Mediterranean diet can reduce inflammation. Therefore, we longitudinally characterized the gut microbiomes of MPN patients in response to Mediterranean or US-style dietary counseling to determine whether there were microbiome-associated changes in inflammation. We did not find significant changes in the gut microbiome associated with diet, but we did find several associations with inflammation. This research paves the way for future studies by identifying potential mechanistic targets implicated in inflammation within the MPN gut microbiome.

microbiology↗

bioRxiv2023

Design of amyloidogenic peptide traps

Segments of proteins with {beta}-strand propensity can self associate to form amyloid fibrils associated with many diseases. These regions often adopt alternative structures in their folded states, or are intrinsically disordered in solution, making it difficult to generate binders or inhibitors with existing strategies. Here we describe a general approach to bind such segments in {beta}-strand and {beta}-hairpin conformations using de novo designed scaffolds that contain deep peptide binding clefts flanked by {beta}-strands that form hydrogen bonds to the peptide upon binding. The designs bind their cognate peptides in vitro with nanomolar affinities and in mammalian cells. The crystal structure of a designed protein-peptide complex is close to the design model, and NMR characterization reveals how the peptide binding cleft is protected in the apo state. We use the approach to design binders to segments of the amyloid forming proteins Transthyretin, Tau, Serum amyloid A1 and A{beta}42. The A{beta} binders block assembly of A{beta} fibrils as effectively as the most potent of the clinically tested antibodies to date.

synthetic biology↗

bioRxiv2022

De novo designed ice-binding proteins from twist-constrained helices

Attaining molecular-level control over solidification processes is a crucial aspect of materials science. To control ice formation, organisms have evolved bewildering arrays of ice-binding proteins (IBPs) but these have poorly understood structure-activity relationships. We propose that reverse engineering using de novo computational protein design can shed light on structureactivity relationships of IBPs. We hypothesized that the model alpha-helical winter flounder antifreeze protein (wfAFP) uses an unusual under-twisting of its alpha-helix to align its putative ice-binding threonine residues in exactly the same direction. We test this hypothesis by designing a series of straight three-helix bundles with an ice-binding helix projecting threonines and two supporting helices constraining the twist of the ice-binding helix. We find that ice recrystallization inhibition by the designed proteins increases with the degree of designed under-twisting, thus validating our hypothesis and opening up new avenues for the computational design of icebinding proteins. Significance StatementIce-binding proteins (IBPs) modulate ice nucleation and growth in cold-adapted organisms so that they can survive in ice-laden environments at (sub)freezing temperatures. The functional repertoire of IBPs is diverse, ranging from inhibition of recrystallization and freezing point depression to shaping of ice crystals and ice nucleation. Precisely how these activities arise from the structure and ice-binding properties of IBPs is poorly understood. We demonstrate through de novo computational protein design that constraining the twist of an ice-binding helix is a key feature determining its ice-binding activity, opening new avenues for the design of synthetic IBPs with activities tailored to the requirements of specific applications, such as cell and tissue cryopreservation.

bioengineering↗

bioRxiv2022

The microbial community dynamics of cocaine sensitization in two behaviorally divergent strains of collaborative cross mice

The gut-brain axis is increasingly recognized as an important pathway involved in cocaine use disorder. Microbial products of the murine gut have been shown to affect striatal gene expression, and depletion of the microbiome by antibiotic treatment alters cocaine-induced behavioral sensitization in C57BL/6J male mice. Some reports suggest that cocaine-induced behavioral sensitization is correlated with drug self-administration behavior in mice. Here we profile the composition of the naive microbiome and its response to cocaine sensitization in two Collaborative Cross (CC) strains. These strains display extremely divergent behavioral responses to cocaine sensitization. A high-responding strain, CC004/TauUncJ (CC04), has a gut microbiome that contains a greater amount of Lactobacillus than the cocaine-nonresponsive strain CC041/TauUncJ (CC41). The gut microbiome of CC41 is characterized by an abundance of Eisenbergella, Robinsonella and Ruminococcus. In response to cocaine, CC04 has an increased Barnsiella population, while the gut microbiome of CC41 displays no significant changes. PICRUSt functional analysis of the functional potential of the gut microbiome in CC04 shows a significant number of potential gut-brain modules altered after exposure to cocaine, specifically those encoding for tryptophan synthesis, glutamine metabolism, and menaquinone synthesis (vitamin K2). Depletion of the microbiome by antibiotic treatment revealed an altered cocaine-sensitization response following antibiotics in female CC04 mice. Depleting the microbiome by antibiotic treatment in males revealed increased infusions for CC04 during a cocaine intravenous self-administration dose-response curve. Together these data suggest that genetic differences in cocaine-related behaviors may involve the microbiome. FUNDINGU01DA043809 to JAB, GMW P50DA039841 to EJC

animal behavior and cognition↗

bioRxiv2022

Accurate Computational Design of 3D Protein Crystals

Protein crystallization plays a central role in structural biology1, with broad impact2 in pharmaceutical formulation3, drug delivery4, biosensing5, and biocatalysis6,7. Despite this importance, the process of protein crystallization remains poorly understood and highly empirical8-10, with largely unpredictable crystal contacts, lattice packing arrangements, and space group preferences, and the programming of protein crystallization through precisely engineered sidechain-sidechain interactions across multiple protein-protein interfaces is an outstanding challenge. Here we develop a general computational approach to designing three-dimensional (3D) protein crystals with pre-specified lattice architectures at atomic accuracy that hierarchically constrains the overall degree of freedoms (DOFs) of the system. We use the approach to design three pairs of oligomers that can be individually purified, and upon mixing, spontaneously self-assemble into large 3D crystals (>100 m). Small-angle X-ray scattering and X-ray crystallography show these crystals are nearly identical to the computational design models, with the design target F4132 and I432 space groups and closely corresponding overall architectures and protein-protein interfaces. The crystal unit cell dimensions can be systematically redesigned while retaining space group symmetry and overall architecture, and the crystals are both extremely porous and highly stable, enabling the robust scaffolding of inorganic nanoparticle arrays. Our approach thus enables the computational design of protein crystals with high accuracy, and since both structure and assembly are encoded in the primary sequence, provides a powerful new platform for biological material engineering.

synthetic biology↗

bioRxiv2022

Therapeutic modulation of the blood-brain barrier and ischemic stroke by a bioengineered FZD4-selective WNT surrogate

Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimers disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/{beta}-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we developed L6-F4-2, a non-lipidated, FZD4-specific surrogate with significantly improved sub-picomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reversed neonatal retinal angiogenesis deficits, but also restored BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduced BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with general applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.

physiology↗

bioRxiv2022

Precisely patterned nanofibers made from extendable protein multiplexes

Molecular systems with coincident cyclic and superhelical symmetry axes have considerable advantages for materials design as they can be readily lengthened or shortened by changing the length of the constituent monomers. Among proteins, alpha helical coiled coils have such symmetric extendable architectures, but are limited by the relatively fixed geometry and flexibility of the helical protomers. Here, we describe a systematic approach to generating modular and rigid repeat protein oligomers with coincident C2 to C8 and superhelical symmetry axes that can be readily extended by repeat propagation. From these building blocks, we demonstrate that a wide range of unbounded fibers can be systematically designed by introducing hydrophilic surface patches that force staggering of the monomers; the geometry of such fibers can be precisely tuned by varying the number of repeat units in the monomer and the placement of the hydrophilic patches.

bioengineering↗

bioRxiv2022

Curation of the Deep Green list of unannotated green lineage proteins to enable structural and functional characterization

An explosion of sequenced genomes and predicted proteomes enabled by low cost deep sequencing has revolutionized biology. Unfortunately, protein functional annotation is more complex, and has not kept pace with the sequencing revolution. We identified unannotated proteins in three model organisms representing distinct parts of the green lineage (Viridiplantae); Arabidopsis thaliana (dicot), Setaria viridis (monocot), and Chlamydomonas reinhardtii (Chlorophyte alga). Using similarity searching we found the subset of unannotated proteins that were conserved between these species and defined them as Deep Green proteins. Informatic, genomic, and structural predictions were leveraged to begin inferring functional information about Deep Green genes and proteins. The Deep Green set was enriched for proteins with predicted chloroplast targeting signals that are predictive of photosynthetic or plastid functions. Strikingly, structural predictions using AlphaFold and comparisons to known structures show that a significant proportion of Deep Green proteins may possess novel protein tertiary structures. The Deep Green genes and proteins provide a starting resource of high value targets for further investigation of potentially new protein structures and functions that are conserved in the green lineage.

plant biology↗

bioRxiv2022

Crystal structure of monkeypox H1 phosphatase, an Antiviral Drug Target

The current monkeypox outbreak has caused over 64,000 global cases, but the effective treatments are very limited. The dual specific phosphatase (H1) from monkeypox antagonizes the immune response and is crucial for viral replication, making it an attractive antiviral target. Here we determined a 1.8-[A] crystal structure of H1, which forms a domain swapped dimer resembling a butterfly. Each active site, which consists of a Cys-Arg-Asp triad, captures a phosphate ion. The observed conformation mimics the final step of catalysis prior to product release. The crystal structure provides a strong foundation for the discovery of new antivirals against this emerging worldwide pathogen.

microbiology↗

bioRxiv2022

Multi-lingual multi-platform investigations of online trade in jaguar parts

We conducted research to understand online trade in jaguar parts and develop tools of utility for jaguars and other species. Our research took place to identify potential trade across 31 online platforms in Spanish, Portuguese, English, Dutch, French, Chinese, and Vietnamese. We identified 230 posts from between 2009 and 2019. We screened the images of animal parts shown in search results to verify if from jaguar; 71 posts on 12 different platforms in four languages were accompanied by images identified as definitely jaguar, including a total of 125 jaguar parts (50.7% posts in Spanish, 25.4% Portuguese, 22.5% Chinese and 1.4% French). Search effort varied among languages due to staff availability. Standardizing for effort across languages by dividing number of posts advertising jaguars by search time and number individual searches completed via term/platform combinations, the adjusted rankings of posts were: Portuguese #1, Chinese 2 (time) & 3 (searches), Spanish 3 & 4; French 5 & 4; English 5 & 2, and Dutch 6. Teeth were most common; 156 posts offered at least 367 apparent teeth. From these, 95 teeth were assessed as definitely jaguar; 71 jaguar teeth could be linked to a location, with the majority of the 71 offered for sale from Mexico, China, Bolivia, and Brazil (26.8, 25.4, 16.9, and 12.7% respectively). Ranking of number of teeth was Mexico (19), China (18), Bolivia (12), Brazil (9), Peru/Ecuador (most accurate probable location) (8), Venezuela (3), Guadeloupe (1), and Uruguay (1). The second most traded item, skins and derivative items were only identified from Latin America: Brazil (7), was followed by Peru (6), Bolivia (3), Mexico (2 and 1 skin piece), and Nicaragua and Venezuela (1 each). Whether by number of posts or pieces, the ranking of parts was teeth, skins/pieces of skins, heads, and bodies. Our research presents a snapshot of online jaguar trade and methods that may have utility for many species now traded online. Our research took place within a longer-term project to assist law enforcement in host countries to better identify potential illegal trade online, with research findings informing hubs in Latin America for building such capacity.

ecology↗