Biology subjects

Jang, S.

Publications and source records attributed to Jang, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Human cell surface-AAV interactomes identify LRP6 as blood-brain-barrier transcytosis receptor and immune cytokine IL3 as AAV9 binder

Adeno-associated viruses (AAVs) are foundational gene delivery tools for basic science and clinical therapeutics. However, lack of mechanistic insight, especially for engineered vectors created by directed evolution, can hamper their application. Here, we adapted an unbiased human cell microarray platform to determine the extracellular and cell surface interactomes of natural and engineered AAVs. We identified a naturally-evolved and serotype-specific interaction between the AAV9 capsid and human interleukin 3 (IL3), with possible roles in host immune modulation, as well as lab-evolved low-density-lipoprotein-receptor-related-protein 6 (LRP6) interactions specific to engineered capsids that cross the blood-brain barrier in non-human primates after intravenous administration. The unbiased cell microarray screening approach also allowed us to identify off-target tissue binding interactions of engineered brain-enriched AAV capsids that may inform vectors peripheral organ tropism and side effects. These results allow confident application of engineered AAVs in diverse organisms and unlock future target-informed engineering of improved viral and non-viral vectors for non-invasive therapeutic delivery to the brain.

bioengineering↗

bioRxiv2023

TolRad: A model for predicting radiation tolerance using Pfam annotations identifies novel radiosensitive bacterial species from reference genomes and MAGs

The trait of ionizing radiation (IR) tolerance is variable between bacterial species, with radiosensitive bacteria succumbing to acute doses around 100Gy and extremophiles able to survive doses exceeding 10,000Gy. While survival screens have identified multiple highly radioresistant bacteria, such systemic searches have not been conducted for radiosensitive bacteria. The taxonomy-level diversity of IR of intolerance across bacteria is poorly understood, as are genetic elements that influence IR sensitivity. Using the protein domain frequencies from 61 bacterial species with experimentally determined IR D10 values (the dose at which only 10% of the population survives) we trained TolRad, a random forest binary classifier, to distinguish between radiosensitive bacteria (D10 < 200Gy) and radiation tolerant bacteria (D10 > 200Gy). On the hidden species, TolRad had an accuracy of 0.900. We applied TolRad to 152 UniProt-hosted bacterial proteomes, including 37 strains from the ATCC Human Microbiome Collection, and classified 34 species as radiosensitive. Whereas IR intolerance (D10 < 200Gy) in the training dataset had been confined to the phylum Proteobacterium, this initial TolRad screen identified radiosensitive bacteria in 2 additional phyla. We experimentally validated the predicted radiosensitivity of a key species of the human microbiome from the Bacteroidota phyla. To demonstrate that TolRad can be applied to Metagenome-Assembled Genome (MAGs), we tested the accuracy of TolRad on Egg-NOG assembled proteomes (0.965) and partial proteomes. Finally, three collections of MAGs were screened using TolRad, identifying further phylum with radiosensitive species and suggesting that environmental conditions influence the abundance of radiosensitive bacteria. ImportanceBacterial species have vast genetic diversity, allowing for life in extreme environments and the conduction of complex chemistry. The ability to harness the full potential of bacterial diversity is hampered by the lack of high-throughput experimental or bioinformatic methods for characterizing bacterial traits. Here, we present a computational model that uses de novo generated genome annotations to classify a bacterium as tolerant of ionizing radiation (IR) or as radiosensitive. This model allows for rapid screening of bacterial communities for low-tolerance species that are of interest for both mechanistic studies into bacterial sensitivity to IR and biomarkers of IR exposure.

bioinformatics↗

bioRxiv2023

Hundreds of antimicrobial peptides create a selective barrier for insect gut symbionts

The spatial organization of gut microbiota is crucial for the functioning of the gut ecosystem, although the mechanisms that organize gut bacterial communities in microhabitats are only partially understood. The gut of the insect Riptortus pedestris has a characteristic microbiota biogeography with a multispecies community in the anterior midgut and a mono-specific bacterial population in the posterior midgut. We show that the posterior midgut region produces massively hundreds of specific antimicrobial peptides (AMPs), the Crypt-specific Cysteine-Rich peptides (CCRs) that have membrane-damaging antimicrobial activity against diverse bacteria but posterior midgut symbionts have elevated resistance. We determined by transposon-sequencing the genetic repertoire in the symbiont Caballeronia insecticola to manage CCR stress, identifying different independent pathways, including novel AMP-resistance pathways unrelated to known membrane homeostasis functions as well as cell envelope functions. Mutants in the corresponding genes have reduced capacity to colonize the posterior midgut, demonstrating that CCRs create a selective barrier and resistance is crucial in gut symbionts. Moreover, once established in the gut, the bacteria differentiate into a CCR-sensitive state, suggesting a second function of the CCR peptide arsenal in protecting the gut epithelia or mediating metabolic exchanges between the host and the gut symbionts. Our study highlights the evolution of an extreme diverse AMP family that contributes to establish and control the gut microbiota.

microbiology↗

bioRxiv2023

Stochastic Modeling of Tinnitus Loudness

There has been no study on the relationship between chronic tinnitus and harmonic templates. Harmonic templates are harmonically structured receptive fields in the auditory system in which all frequency components are integer multiples of a common fundamental frequency (F0). In this study, data from 19 harmonic templates from each of 196 chronic tinnitus patients were analyzed and mathematical modeling was performed to quantify the loudness of chronic tinnitus. High-resolution hearing threshold data were obtained by algorithmic pure tone audiometry (PTA) conducting automated PTA at 134 frequency bands with 1/24 octave resolution from 250 Hz to 12,000 Hz. The result showed that there is an intriguing relationship between the auditory instability of harmonic templates and simplified tinnitus severity score (STSS). This study provides several mathematical models to estimate tinnitus severity and the precise quantification of the loudness of chronic tinnitus. Our computational models and analysis of the behavioral hearing threshold fine structure suggest that the cause of severe chronic tinnitus could be a severe disparity between different temporal capacities of each neural oscillator in a certain harmonic template.

neuroscience↗

bioRxiv2022

An RNA interference (RNAi) toolkit and its utility for functional genetic analysis of Leishmania ( Viannia )

RNA interference (RNAi) is a powerful tool whose efficacy against a broad range of targets enables functional genetic tests individually or systematically. However, the RNAi pathway has been lost in evolution by a variety of eukaryotes including most Leishmania sp. RNAi was retained in species of the Leishmania subgenus Viannia, and here we describe the development, optimization, and application of RNAi tools to the study of L. (Viannia) braziliensis. We developed vectors facilitating generation of long-hairpin or "stem-loop" (StL) RNAi knockdown constructs, using Gateway site-specific recombinase technology. A survey of applications of RNAi in L. braziliensis included genes interspersed within multigene tandem arrays such as QDPR, a potential target or modulator of antifolate sensitivity. Other tests include genes involved in cell differentiation and amastigote proliferation (A600), and essential genes of the intraflagellar transport (IFT) pathway. We tested a range of stem lengths targeting the L. braziliensis hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and reporter firefly luciferase (LUC) genes and found that the efficacy of RNAi increased with stem length, and fell off greatly below about 128 nt. We used the StL length dependency to establish a useful hypomorphic approach not possible with other gene ablation strategies, with shorter IFT140 stems yielding viable cells with compromised flagellar morphology. We showed that co-selection for RNAi against adenine phosphoryl transferase (APRT1) using 4-aminopyrazolpyrimidine (APP) could increase the efficacy of RNAi against reporter constructs, a useful tool that may facilitate improvements in future work. Thus, for many genes, RNAi provides a useful tool for studying Leishmania gene function with some unique advantages.

microbiology↗

bioRxiv2022

Using large-scale whole-genome sequence data for single-step genomic predictions in maternal and terminal pig lines

BackgroundWhole-genome sequence (WGS) data harbor causative variants that may not be present in the regular SNP chip data. The objective of this study was to investigate the impact of using preselected variants from WGS for single-step genomic predictions in maternal and terminal pig lines with up to 1.8k sequenced and 104k imputed sequenced animals per line. MethodsTwo maternal and four terminal lines were investigated for eight and seven traits, respectively. The number of sequenced animals ranged from 1,365 to 1,491 in maternal lines and 381 to 1,865 in terminal lines. Imputation occurred within each line, and the number of animals imputed to sequence ranged from 66k to 76k in maternal lines and 29k to 104k in terminal lines. Two preselected SNP sets were generated based on genome-wide association study (GWAS). Top40k included the SNP with the lowest p-value in each of 40k genomic windows; ChipPlusSign included significant variants integrated into the regular porcine SNP chip. Single-step genomic predictions with equal or different SNP variances using those SNP sets were compared to the regular porcine SNP chip. ResultsIn maternal lines, ChipPlusSign, and Top40k showed, on average, 0.62%, and 4.9% increased accuracy compared to the regular porcine SNP chip. The greatest changes were for fertility traits with Top40k, where the initial accuracy based on the SNP chip was low. However, for terminal lines, Top40k resulted in a loss of accuracy of 1% on average. Only ChipPlusSign provided a positive, albeit small, gain (0.85%). Assigning different variances for SNP slightly improved accuracies when using variances obtained from BayesR; however, the increase was inconsistent across the lines and traits. ConclusionsThe benefit of using sequence data depends on the line, size of the genotyped population, and how the WGS variants are preselected. When WGS is available on hundreds of thousands of animals, the advantage of sequence data is present but limited in maternal and terminal pig lines.

genomics↗

bioRxiv2022

Genome-scale CRISPR screening reveals host factors required for ribosome formation and viral replication

Viruses are known to co-opt host machinery for translation initiation, but less is known about which host factors are required for the formation of ribosomes used to synthesize viral proteins. Using a loss of function CRISPR screen, we show that synthesis of a flavivirus encoded reporter depends on multiple host factors, including several 60S ribosome biogenesis proteins. Viral phenotyping revealed that two of these factors, SBDS, a known ribosome biogenesis factor, and the relatively uncharacterized protein SPATA5, were broadly required for replication of flaviviruses, coronaviruses, alphaviruses, paramyxoviruses, an enterovirus, and a poxvirus. Mechanistic studies revealed that loss of SPATA5 caused defects in ribosomal RNA processing and ribosome assembly, suggesting that this human protein may be a functional ortholog of yeast Drg1. These studies implicate specific ribosome biogenesis proteins as viral host dependency factors that are required for synthesis of virally encoded protein and accordingly, optimal viral replication.

microbiology↗

bioRxiv2022

Human-specific progenitor sub-domain contributes to extended neurogenesis and increased motor neuron production

Neurogenesis lasts ~10 times longer in developing humans compared to mice, resulting in >1000-fold more neurons in the human central nervous system. Expansion of human neocortex has been in part attributed to the population of outer radial glia and amplifying progenitors that increase the output of neurogenic lineages. However, outer radial glia appear to be absent in many regions of the developing human nervous system, prompting us to search for alternative populations of progenitors that contribute to the expansion of human neurogenesis in one such region - the developing spinal cord. To this end, we performed high-temporal resolution single-cell expression analysis of human and mouse motor neuron progenitors generated from pluripotent stem cells in vitro. Alignment of human and mouse data using canonical correlation analysis identified "human-specific" progenitor clusters characterized by early co-expression of NKX2-2 and OLIG2 that lacked an orthologous murine counterpart. A matching progenitor population has been previously described in the human embryonic spinal cord1, but its function remained unknown. Our lineage tracing analysis demonstrates that these cells function as ventral motor neuron progenitors (vpMNs), but in contrast to classical pMNs, vpMNs exhibit increased Notch activity and generate motor neurons in a delayed and protracted manner. Concomitantly, vpMNs undergo more rounds of cell division before undergoing neurogenesis, leading to ~2-fold increase in total motor neuron output, and contributing preferentially to later-born, limb-innervating motor neuron subtypes. Thus, instead of relying on transit-amplifying progenitors, human spinal cord evolved a novel progenitor subdomain that extends timescales and expands output of human motor neurogenesis.

developmental biology↗

bioRxiv2022

DBlink: Dynamic localization microscopy in super spatiotemporal resolution via deep learning

Single molecule localization microscopy (SMLM) has revolutionized biological imaging, improving the spatial resolution of traditional microscopes by an order of magnitude. However, SMLM techniques depend on accumulation of many localizations over thousands of recorded frames to yield a single super-resolved image, which is time consuming. Hence, the capability of SMLM to observe dynamics has always been limited. Typically, a few minutes of data acquisition are needed to reconstruct a single super-resolved frame. In this work, we present DBlink, a novel deep-learning-based algorithm for super spatiotemporal resolution reconstruction from SMLM data. The input to DBlink is a recorded video of single molecule localization microscopy data and the output is a super spatiotemporal resolution video reconstruction. We use bi-directional long short term memory (LSTM) network architecture, designed for capturing long term dependencies between different input frames. We demonstrate DBlink performance on simulated data of random filaments and mitochondria-like structures, on experimental SMLM data in controlled motion conditions, and finally on live cell dynamic SMLM. Our neural network based spatiotemporal interpolation method constitutes a significant advance in super-resolution imaging of dynamic processes in live cells.

bioengineering↗

bioRxiv2022

Dimensionality of genomic information and its impact on GWA and variant selection: a simulation study

BackgroundIdentifying true-positive variants in genome-wide associations (GWA) depends on several factors, including the number of genotyped individuals. The limited dimensionality of the genomic information may give insights into the optimal number of individuals to use in GWA. This study investigated different discovery set sizes in GWA based on the number of largest eigenvalues explaining a certain proportion of variance in the genomic relationship matrix (G). An additional investigation included the change in accuracy by adding variants, selected based on different set sizes, to the regular SNP chips used for genomic prediction. MethodsSequence data were simulated containing 500k SNP with 200 or 2000 quantitative trait nucleotides (QTN). A regular 50k panel included one every ten simulated SNP. Effective population size (Ne) was 20 and 200. The GWA was performed with the number of genotyped animals equivalent to the number of largest eigenvalues of G (EIG) explaining 50, 60, 70, 80, 90, 95, 98, and 99% of the variance. In addition, the largest discovery set consisted of 30k genotyped animals. Limited or extensive phenotypic information was mimicked by changing the trait heritability. Significant and high effect size SNP were added to the 50k panel and used for single-step GBLUP with and without weights. ResultsUsing the number of genotyped animals corresponding to at least EIG98 enabled the identification of QTN with the largest effect sizes when Ne was large. Smaller populations required more than EIG98. Furthermore, using genotyped animals with higher reliability (i.e., higher trait heritability) helped better identify the most informative QTN. The greatest prediction accuracy was obtained when the significant or the high effect SNP representing twice the number of simulated QTN were added to the 50k panel. Weighting SNP differently did not increase prediction accuracy, mainly because of the size of the genotyped population. ConclusionsAccurately identifying causative variants from sequence data depends on the effective population size and, therefore, the dimensionality of genomic information. This dimensionality can help identify the suitable sample size for GWA and could be considered for variant selection. Even when variants are accurately identified, their inclusion in prediction models has limited implications.

genomics↗

bioRxiv2022

PD1+ T-cells correlate with Nerve Fiber Density as a prognostic biomarker in patients with resected perihilar cholangiocarcinoma.

Background & AimsPerihilar cholangiocarcinoma (pCCA) is a rare hepatobiliary malignancy. Nerve fiber invasion (NFI) shows cancer invading the nerve and is considered an aggressive feature. Nerve fiber density (NFD) consists of small nerve fibers without cancer invasion and is divided into high NFD (high numbers of small nerve fibers) or low NFD (low numbers of small nerve fibers). We aim to explore differences in immune cell populations and survival. MethodsWe applied multiplex immunofluorescence (mIF) on 47 pCCA surgically resected patients and investigated the immune cell composition in the tumor microenvironment (TME) of different nerve fiber phenotypes (NFI, high and low NFD). Extensive group comparison was carried out and the association with overall survival (OS) was assessed. ResultsThe NFI ROI was measured with highest CD68+ macrophage levels among 3 ROIs (NFI compared to tumor free p= 0.016 and to tumor p=0.034). Further, for NFI patients the density of co-inhibitory markers CD8+PD1+ and CD68+PD1+ were more abundant in the tumor rather than NFI ROI (p= 0.004 and p= 0.0029 respectively). Comparison between patients with NFD and NFI groups, the signals of co-expression of CD8+PD1+ as well as CD68+PD1+ were significantly higher in the high NFD group (p= 0.027 and p= 0.044, respectively). The OS for high NFD patients was 92 months median OS (95% CI:41-142), for low NFD patients 20 months ((95% CI: 4-36) and for NFI group of patients 19 months (95% CI 7-33). The OS for high NFD patients was significantly better compared to low NFD (p= 0.046) and NFI (p= 0.032). ConclusionsPD1+ T-cells correlate with high NFD as a prognostic biomarker, the biological pathway behind this needs to be investigated. Lay summaryNerve fibers play a dual role in the tumor microenvironment in pCCA. In our previous study, we showed that the presence of high numbers of small nerve fibers is associated with a better overall survival. In addition, we found that in high NFD patients PD1+ T-cells are significantly overexpressed. Therefore, we present high NFD as a promising prognostic biomarker. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/475344v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@e30fadorg.highwire.dtl.DTLVardef@11a4e4aorg.highwire.dtl.DTLVardef@a04d8org.highwire.dtl.DTLVardef@1c425db_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Crosstalk between Fat Mass and Obesity-related (FTO) and multiple WNT signaling pathways

Fat Mass and Obesity-related (FTO) gene is associated with a diverse set of human diseases. Yet, the functional landscape of FTO remains largely unknown, most likely owing to its wide range of mechanistic roles and cell-type-specific targets. Here, we discover the intricate role of FTO in multiple WNT signaling pathways. Re-analyses of public data identified the bifurcation of canonical and noncanonical WNT pathways as the major role of FTO. In FTO-depleted cells, we find that the canonical WNT/{beta}-Catenin signaling is inhibited in a non-cell autonomous manner via the upregulation of DKK1. Simultaneously, this upregulation of DKK1 promotes cell migration via activating the noncanonical WNT/PCP pathway. Unexpectedly, we also find that the canonical WNT/STOP signaling induces the accumulation of cytoplasmic FTO proteins. This subsequently leads to the stabilization of mRNAs via RNA demethylation, revealing a previously uncharacterized mode of WNT action in RNA regulation. Altogether, this study places the functional context of FTO at the branching point of multiple WNT signaling pathways which may explain the wide spectrum of FTO functions.

molecular biology↗

bioRxiv2021

RNA splicing programs define tissue compartments and cell types at single cell resolution

More than 95% of human genes are alternatively spliced. Yet, the extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach that is agnostic to transcript annotation, to detect cell-type-specific regulated splicing in > 110K carefully annotated single cells from 12 human tissues. Using 10x data for discovery, 9.1% of genes with computable SpliZ scores are cell-type specifically spliced. These results are validated with RNA FISH, single cell PCR, and in high throughput with Smart-seq2. Regulated splicing is found in ubiquitously expressed genes such as actin light chain subunit MYL6 and ribosomal protein RPS24, which has an epithelial-specific microexon. 13% of the statistically most variable splice sites in cell-type specifically regulated genes are also most variable in mouse lemur or mouse. SpliZ analysis further reveals 170 genes with regulated splicing during sperm development using, 10 of which are conserved in mouse and mouse lemur. The statistical properties of the SpliZ allow model-based identification of subpopulations within otherwise indistinguishable cells based on gene expression, illustrated by subpopulations of classical monocytes with stereotyped splicing, including an un-annotated exon, in SAT1, a Diamine acetyltransferase. Together, this unsupervised and annotation-free analysis of differential splicing in ultra high throughput droplet-based sequencing of human cells across multiple organs establishes splicing is regulated cell-type-specifically independent of gene expression.

bioinformatics↗

bioRxiv2020

Targeting CSF-1 ameliorates experimental autoimmune encephalomyelitis by depleting inflammatory monocytes and microglia in the central nervous system without affecting quiescent microglia.

The receptor for colony stimulating factor 1 (CSF-1R) is important for the survival and function of myeloid cells that mediate pathology during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). CSF-1 and IL-34, the ligands of CSF-1R, have similar bioactivities but distinct tissue and context-dependent expression patterns, suggesting that they have different roles. This could be the case in EAE, given that CSF-1 expression is upregulated in the CNS, while IL-34 remains constitutively expressed. We found that targeting CSF-1 with neutralizing mAb halted ongoing EAE, with efficacy superior to CSF-1R inhibitor BLZ945, whereas IL-34 neutralization had no effect, suggesting that pathogenic myeloid cells were maintained by CSF-1, not IL-34. Both anti-CSF-1- and BLZ945-treatment greatly reduced numbers of monocyte-derived cells and microglia in the CNS. However, anti-CSF-1 selectively depleted inflammatory microglia and monocytes in inflamed CNS areas, whereas BLZ945 depleted virtually all myeloid cells, including quiescent microglia, throughout the CNS. Anti-CSF-1 treatments reduced the size of demyelinated lesions, and microglial activation in the grey matter. Lastly, we found that bone marrow-derived immune cells were the major mediators of CSF-1R-dependent pathology, while microglia played a lesser role. Our findings suggest that targeting CSF-1 could be effective in ameliorating MS pathology, while preserving the homeostatic functions of myeloid cells, thereby minimizing risks associated with total ablation of CSF-1R-dependent cells. Significance StatementMultiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases characterized by accumulation of myeloid immune cells into the central nervous system (CNS). Both harmful and beneficial myeloid cells are present in EAE/MS, and a goal of MS therapy is to preferentially remove harmful myeloid cells. The receptor for CSF-1 (CSF-1R) is found on myeloid cells and it is important for their survival. CSF-1R can bind two ligands, CSF-1 and IL-34, but is unknown whether their functions in EAE/MS differ. We found that blocking CSF-1 depleted only harmful myeloid cells in the CNS and suppressed EAE, whereas blocking IL-34 had no effect. Thus, we propose that blocking CSF-1 could be a novel therapy for MS.

immunology↗

bioRxiv2020

A DNA-origami nuclear pore mimic reveals nuclear entry mechanisms of HIV-1 capsid

The capsid of human immunodeficiency virus 1 (HIV-1) plays a pivotal role in viral nuclear import, but the mechanism by which the viral core passages the nuclear pore complex (NPC) is poorly understood. Here, we use DNA-origami mimics of the NPC, termed NuPODs (NucleoPorins Organized by DNA), to reveal the mechanistic underpinnings of HIV-1 capsid nuclear entry. We found that trimeric interface formed via three capsid protein hexamers is targeted by a triple-arginine (RRR) motif but not the canonical phenylalanine-glycine (FG) motif of NUP153. As NUP153 is located on the nuclear face of the NPC, this result implies that the assembled capsid must cross the NPC in vivo. This hypothesis is corroborated by our observations of tubular capsid assemblies penetrating through NUP153 NuPODs. NUP153 prefers to bind highly curved capsid assemblies including those found at the tips of viral cores, thereby facilitating capsid insertion into the NPC. Furthermore, a balance of capsid stabilization by NUP153 and deformation by CPSF6, along with other cellular factors, may allow for the intact capsid to pass NPCs of various sizes. The NuPOD system serves as a unique tool for unraveling the previously elusive mechanisms of nuclear import of HIV-1 and other viruses.

microbiology↗

bioRxiv2020

Signature of N-terminal domain (NTD) structural re-orientation in NPC1 for proper alignment of cholesterol transport: Molecular dynamics study with mutation

The lysosomal membrane protein NPC1 (Niemann-Pick type C1) and NPC2 (Niemann-Pick type C2) are main players of cholesterol control in lysosome and it is known that mutation on these proteins leads to cholesterol trafficking related disease, called Niemann-Pick disease type C (NPC) disease. The mutation R518W or R518Q on NPC1 is one of such disease-related mutations, causing reduced cholesterol transport by half, resulting in accumulation of cholesterol and lipids in late endosomal/lysosomal region of the cell. Even though there has been significant progress in understanding cholesterol transport by NPC1 in combination with NPC2, especially after the structural determination of full length NPC1 in 2016, many details such as interaction of full length NPC1 with NPC2, molecular motions responsible for cholesterol transport during and after this interaction, and structure and function relations of many mutations are still not well understood. We report the extensive molecular dynamics simulations to gain insight into the structure and motions of NPC1 lumenal domain for cholesterol transport and disease behind the mutation (R518W). It is found that the mutation induces structural shift of NTD (N-terminal domain), toward the loop region in MLD (middle lumenal domain), which is believed to play central role in interaction with NPC2 protein, such that the interaction with NPC2 protein might be less favorable compare to wild NPC1. Also, the simulation indicates the possible re-orientation of the NTD, aligning to form an internal tunnel, after receiving the cholesterol from NPC2 with wild NPC1 unlike the mutated one, a possible pose for further action in cholesterol trafficking. We believe the current study can provide better understanding on the cholesterol transport by NPC1, especially the role of NTD of NPC1, in combination with NPC2 interaction. Synopsismodeling study of cholesterol binding protein NPC1

biophysics↗

bioRxiv2020

Cytoplasmic CPSF6 regulates HIV-1 capsid trafficking and infection in a cyclophilin A-dependent manner

Human immunodeficiency virus type 1 (HIV-1) capsid binds host proteins during infection, including cleavage and polyadenylation specificity factor 6 (CPSF6) and cyclophilin A (CypA). We observe that HIV-1 infection induces higher-order CPSF6 formation and capsid-CPSF6 complexes co-traffic on microtubules. CPSF6-capsid complex trafficking is impacted by capsid alterations that reduce CPSF6 binding or by excess cytoplasmic CPSF6 expression, both of which are associated with decreased HIV-1 infection. Higher-order CPSF6 complexes bind and disrupt HIV-1 capsid assemblies in vitro. Disruption of HIV-1 capsid binding to CypA leads to increased CPSF6 binding and altered capsid trafficking, resulting in reduced infectivity. Our data reveal an interplay between CPSF6 and CypA that is important for cytoplasmic capsid trafficking and HIV-1 infection. We propose that CypA prevents HIV-1 capsid from prematurely engaging cytoplasmic CPSF6 and that differences in CypA cellular localization and innate immunity may explain cell-specific variations in HIV-1 capsid trafficking and uncoating. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/136697v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@646829org.highwire.dtl.DTLVardef@1f25cf9org.highwire.dtl.DTLVardef@b5e696org.highwire.dtl.DTLVardef@3593a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

ThiL is a valid antibacterial target that is essential for both thiamine biosynthesis and salvage pathway in Pseudomonas aeruginosa

Thiamine pyrophosphate (TPP) is an essential cofactor for various pivotal cellular processes in all living organisms, including bacteria. As thiamine biosynthesis occurs in bacteria but not humans, bacterial thiamine biosynthesis is an attractive target for antibiotic development. Among enzymes in the thiamine biosynthetic pathway, thiamine monophosphate kinase (ThiL) catalyzes the final step of the pathway, phosphorylating thiamine monophosphate (TMP) to produce TPP. In this work, we extensively investigated ThiL in Pseudomonas aeruginosa, a major pathogen of hospital-acquired infections. We demonstrated that thiL deletion abolishes not only thiamine biosynthesis but also thiamine salvage capability, showing growth defects of the {Delta}thiL mutant even in the presence of thiamine derivatives except TPP. Most importantly, the pathogenesis of the {Delta}thiL mutant was markedly attenuated compared to wild-type bacteria, with lower inflammatory cytokine induction and 103~104 times decreased bacterial load in an in vivo infection model where the intracellular TPP level is in the submicromolar range. In order to validate P. aeruginosa ThiL (PaThiL) as a new drug target, we further characterized its biochemical properties determining a Vmax of 4.0{+/-}0.2 nomol{middle dot}min-1 and KM values of 111{+/-}8 and 8.0{+/-}3.5M for ATP and TMP, respectively. A subsequent in vitro small molecule screening identified PaThiL inhibitors including WAY213613 that is a noncompetitive inhibitor with a Ki value of 13.4{+/-}2.3 M and a potential antibacterial activity against P. aeruginosa. This study proved that PaThiL is a new drug target against P. aeruginosa providing comprehensive biological and biochemical data that could facilitate to develop a new repertoire of antibiotics.

microbiology↗