Biology subjects

Alam, A.

Publications and source records attributed to Alam, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Prime Editing Corrects the HBB Codon 8/9 (+G) Mutation in Patient-Derived Induced Pluripotent Stem Cells and Restores β-Globin Expression in iPSC-Derived Erythroid Cells

Homozygosity for the HBB codon 8/9 (+G) frameshift (c.27dup (p.Ser10ValfsTer14)) causes transfusion-dependent {beta}-thalassaemia and is common in South Asia. Prime editing can reverse this insertion without double-strand breaks or donor DNA, but its efficiency depends on pegRNA design. We derived Sendai-reprogrammed iPSCs from a homozygous patient, optimised PEmax editing (spacer, pegRNA extension, secondary nick, MLH1dn) and compared patient, PEmax-treated and control iPSC-derived erythroid cells by flow cytometry, colony assays, RT-qPCR, western blotting and cation-exchange HPLC. Patient iPSCs had a normal 46,XY karyotype, expressed pluripotency markers, formed all three germ layers and were Sendai-free by passage 15. A PAM-disrupting spacer with a +68 nicking sgRNA gave the highest intended-edit frequency 8.4%; MLH1dn added little. PEmax-treated cultures matured and formed colonies more like control than patient cultures, restored HBB transcript (about 4-fold control iPSC-derived cells) and detectable {beta}-globin protein, and contained an HPLC fraction consistent with HbA ( undetectable in patient cells). Fetal haemoglobin (about 80%) and embryonic globin remained predominant. Prime editing corrects HBB c.27dupG in patient iPSCs and restores {beta}-globin expression, with partial restoration of adult haemoglobin in a fetal/embryonic-type erythroid background. Validation in additional donors, haematopoietic stem cells and in vivo is required.

cell biology↗

bioRxiv2026

Single-cell profiling reveals disruption of cell-cycle homeostasis following JDP2 depletion in colorectal epithelial cells

Intestinal epithelial cells (IECs) undergo rapid and continuous renewal to maintain gut epithelial homeostasis and barrier integrity at the interface with luminal microbiota, dietary antigens, and enteric pathogens. IEC proliferation is tightly regulated by a complex machinery of cell cycle regulators and effectors, and genetic or transcriptional dysregulation of cell-cycle regulatory pathways is a hallmark of colorectal cancer development and progression. Previously, JDP2 (Jun dimerization protein 2) has been shown to be involved in cell cycle control and is associated with an array of cancers. JDP2 functions as a transcription factor or epigenetic regulator depending on cellular context, disease, and cancer types. However, the role of JDP2 in CRC, cell cycle homeostasis of colon cancer cells, and their fate remains poorly defined. In this study, we found that patients with higher JDP2 expression have significantly worse survival than patients with lower JDP2 expression. Our functional assays showed that JDP2 depletion increased EdU incorporation during S phase and PHH3 abundance, which is consistent with increased proliferative and mitotic activity. Our single-cell RNA sequencing and transcriptomic profiling further revealed reprogramming of cell-cycle-associated transcriptional states following JDP2 depletion. Differential-expression and pathway analysis identified changes in mitotic and cell-cycle regulatory genes, including programs involving cyclins, cyclin-dependent kinases, chromosome segregation, and mitotic progression. We also found that lower residual JDP2 expression was associated with escalated G2/M representation and a change toward later states along a G1 - S - G2/M-associated transcriptional trajectory. Furthermore, low-JDP2 cells showed significantly greater late-state occupancy than High-JDP2 cells (33.6% versus 24.0%; paired P=0.048). Among the JDP2-siRNA-treated cells, analysis of residual JDP2-expressing cells also revealed heterogeneous associations of JDP2 with cell-cycle, survival, apoptotic, and stress-response programs, consistent with JDP2's context-dependent transcriptional functions. Together, these data identify JDP2 as an important component of intestinal epithelial cell-cycle homeostasis and transcriptional landscape and suggest that JDP2 depletion perturbs the balance of proliferative cell states.

molecular biology↗

bioRxiv2026

Campylobacter jejuni Infection Is Associated With Cell-Cycle, Redox, and Metabolic Remodeling of Human Intestinal Epithelial Cells at Single-Cell Resolution

Campylobacter jejuni is the leading cause of bacterial enteric infections worldwide, including in the US. It is also a zoonotic pathogen that is transmitted by food and water, causing diarrhea and intestinal inflammation, and is responsible for high morbidity and mortality in young children, the elderly, and immunocompromised patients. During infection, C. jejuni profoundly perturbs intestinal epithelial physiology, leading to widespread mucosal damage and inflammatory response. However, the heterogeneity and coordination of host transcriptional responses remain incompletely defined. We performed single-cell RNA sequencing of Caco-2 intestinal epithelial cells from uninfected Control and C. jejuni-infected biological samples using 10x Genomics GEM-X Flex chemistry. Our dataset comprised 159,088 cells and 18,142 measured features. A Seurat workflow resolved 19 transcriptional states and revealed a highly reproducible condition-associated redistribution of clusters across biological replicates. Our study demonstrated that C. jejuni infection was associated with increased representation of G2/M-classified cells and coordinated induction of checkpoint and mitotic-spindle genes, including CDKN1A, WEE1, MAD2L1, BUB1B, PLK1, CDC20, CDK1, and UBE2C. Furthermore, ranked enrichment independently identified a C. jejuni-enriched Reactome mitotic spindle checkpoint program (NES ~1.67, FDR ~0.013), while Hallmark analysis identified strong TNF/NF-kB, hypoxia, G2M checkpoint, TGF-beta, glycolysis, apoptosis, p53, and mTORC1-associated programs. Infection was also associated with significantly decreased JDP2 and higher NOX1 and DUOX2 gene expression. Furthermore, scMetabolism/AUCell identified 20 KEGG metabolic pathways at FDR <0.05 in a balanced 10,000-cell analysis, with 12 lower and 8 higher in infection. Together, these data support a model in which C. jejuni dramatically restructures epithelial transcriptional states and is accompanied by mitotic-checkpoint, inflammatory/stress, redox, and metabolic remodeling.

microbiology↗

bioRxiv2025

Mechanisms involved in the lipid membrane tubulation activity of the pH-responsive MakA protein from Vibrio cholerae

Bacterial pore-forming toxins (PFTs) are key virulence factors that disrupt host cell membranes. The Vibrio cholerae cytotoxin MakA, a member of the ClyA structural family of -PFTs, exhibits a unique, pH-dependent ability to induce lipid membrane tubulation. This study elucidates the molecular mechanisms underlying this process. We identify the N-terminal histidine residue (His30) of MakA as a critical pH sensor that is indispensable for membrane tubulation but not required for membrane binding and cell lysis. Substitution of His30 with lysine, MakAH30K , abrogated tubulation without affecting membrane insertion, instead triggering aberrant membrane fusion events. Circular dichroism spectroscopy analysis revealed that the MakAH30K substitution attenuates the robust -helical transition observed in wild-type MakA upon lipid membrane binding, providing a molecular explanation for its functional defect. Furthermore, we established that membrane cholesterol contributes to MakA-induced tubulation of lipid membranes, as its absence or mutation of a putative cholesterol-binding motif MakAI236D&I237D abolishes both membrane association and tubule formation. A key implication of our work is the mechanistic uncoupling of membrane tubulation from cytolysis, revealing that MakA can perturb membranes via distinct pathways. These findings define a sophisticated activation mechanism where His30 protonation in the acidic host environment and specific cholesterol binding synergistically drive the conformational change necessary for helical oligomerization and membrane tubulation. We discuss our findings from the viewpoint of possible convergent evolution, where bacterial PFTs like MakA partly may mimic ancient membrane remodelers involved in membrane repair pathways, such as ESCRT-III, but for bacterial fitness in host interactions and pathogenesis. Moreover, the present study provides a foundation for possible development of therapeutic strategies aimed at inhibiting these critical interactions.

biochemistry↗

bioRxiv2025

Pro-restitutive Bacteroides thetaiotaomicron reprograms the transcriptome of intestinal epithelial cells by modulating the expression of genes essential for proliferation and migration.

The mammalian intestine harbors a highly complex, very diverse, and numerically vast community of symbiotic microorganisms, which profoundly influence the development and maintenance of the intestinal barrier function. Alterations in microbial composition, known as dysbiosis, are observed in Inflammatory Bowel Disease (IBD), colorectal cancer (CRC), and gastrointestinal infections; however, the exact causal relationship between these changes and the resolution of intestinal inflammation and the repair of damaged mucosa remains unclear. Notably, IBD is not only marked by dysbiosis but also by changes in microbial metabolic pathways and metabolite landscape in the intestinal lumen. The small molecules and microbial metabolites present in the intestinal lumen have emerged as potential regulators of gut pathology, cancer, and mucosal repair. Investigating how altered microbiota and microbial metabolic activities influence intestinal epithelial cells (IEC) can provide insights into their role in the regeneration of mucosal epithelia and restoration of gut barrier functions. This knowledge can be harnessed to promote intestinal homeostasis, prevent relapse, and prolong remission of IBD. To dissect the complex interplay between the gut microbiome and IEC, we focused on the overrepresented bacterium Bacteroides thetaiotaomicron. Here, we show that B. thetaiotaomicron and Akkermansia muciniphila, the dominant members of gut microbiota, expand during the repair & resolution phase of the chemically induced acute murine colitis. Furthermore, our bioinformatics analysis demonstrated that the elevated relative abundance of B. thetaiotamicron was also accompanied by rewiring of bacterial metabolic programs towards the essential amino acid metabolism, polyamine synthesis and utilization, stress response mechanisms, cell envelope biogenesis, and nutrient scavenging. Our RNA sequencing and transcriptomic analysis of primary human colonic epithelial cells cocultured with B. thetaiotaomicron showed that B. thetaiotaomicron stimulates the expression of genes and pathways involved in different cellular functions, including proliferation, differentiation, adhesion, lipid metabolism, migration, chemotaxis, and receptor expression. Our study emphasizes the crucial functions of the gut microbiome and metabolic activities in regulating the functions of intestinal epithelial cells during the repair of injured gut mucosa. Thus, these microorganisms and their metabolism hold promise as potential therapeutic agents.

microbiology↗

bioRxiv2024

Structural insight into binding site access and ligand recognition by human ABCB1

ABCB1 is a broad-spectrum efflux pump central to cellular drug handling and multidrug resistance in humans. However, its mechanisms of poly-specific substrate recognition and transport remain poorly resolved. Here we present cryo-EM structures of lipid embedded human ABCB1 in its apo, substrate-bound, inhibitor-bound, and nucleotide-trapped states at 3.4-3.9 [A] resolution without using stabilizing antibodies or mutations and each revealing a distinct conformation. The substrate binding site is located within one half of the molecule and, in the apo state, is obstructed by transmembrane helix (TM) 4. Substrate and inhibitor binding are distinguished by major differences in TM arrangement and ligand binding chemistry, with TM4 playing a central role in all conformational transitions. Our data offer fundamental new insights into the role structural asymmetry, secondary structure breaks, and lipid interactions play in ABCB1 function and have far-reaching implications for ABCB1 inhibitor design and predicting its substrate binding profiles.

biochemistry↗

bioRxiv2024

Microbial Metabolite Trimethylamine N-Oxide Promotes Campylobacter jejuni Infection by Escalating Intestinal Inflammation, Epithelial Damage, and Barrier Disruption

The interactions between Campylobacter jejuni, a critical foodborne cause of gastroenteritis, and the intestinal microbiota during infection are not completely understood. The crosstalk between C. jejuni and its host is impacted by the gut microbiota through mechanisms of competitive exclusion, microbial metabolites, or immune response. To investigate the role of gut microbiota on C. jejuni pathogenesis, we examined campylobacteriosis in the IL10KO mouse model, which was characterized by an increase in the relative abundance of intestinal proteobacteria, E. coli, and inflammatory cytokines during C. jejuni infection. We also found a significantly increased abundance of microbial metabolite Trimethylamine N-Oxide (TMAO) in the colonic lumens of IL10KO mice. We further investigated the effects of TMAO on C. jejuni pathogenesis. We determined that C. jejuni senses TMAO as a chemoattractant and the administration of TMAO promotes C. jejuni invasion into Caco-2 monolayers. TMAO also increased the transmigration of C. jejuni across polarized monolayers of Caco-2 cells, decreased TEER, and increased C. jejuni-mediated intestinal barrier damage. Interestingly, TMAO treatment and presence during C. jejuni infection of Caco-2 cells synergistically caused an increased inflammatory cytokine expression, specifically IL-1{beta} and IL-8. These results establish that C. jejuni utilizes microbial metabolite TMAO for increased virulence during infection.

microbiology↗

bioRxiv2024

A Functional Map of the Human Intrinsically Disordered Proteome

Intrinsically disordered regions (IDRs) represent at least one-third of the human proteome and defy the established structure-function paradigm. Because IDRs often have limited positional sequence conservation, the functional classification of IDRs using standard bioinformatics is generally not possible. Here, we show that evolutionarily conserved molecular features of the intrinsically disordered human proteome (IDR-ome), termed evolutionary signatures, enable classification and prediction of IDR functions. Hierarchical clustering of the human IDR-ome based on evolutionary signatures reveals strong enrichments for frequently studied functions of IDRs in transcription and RNA processing, as well as diverse, rarely studied functions, ranging from sub-cellular localization and biomolecular condensates to cellular signaling, transmembrane transport, and the constitution of the cytoskeleton. We exploit the information that is encoded within evolutionary conservation of molecular features to propose functional annotations for every IDR in the human proteome, inspect the conserved molecular features that correlate with different functions, and discover frequently co-occurring IDR functions on the proteome scale. Further, we identify patterns of evolutionary conserved molecular features of IDRs within proteins of unknown function and disease-risk genes for conditions such as cancer and developmental disorders. Our map of the human IDR-ome should be a valuable resource that aids in the discovery of new IDR biology.

bioinformatics↗

bioRxiv2024

Enhanced mucosal mitochondrial function corrects dysbiosis and OXPHOS metabolism in IBD

BackgroundMitochondrial (Mito) dysfunction in IBD reduces mucosal O2 consumption and increases O2 delivery to the microbiome. Increased enteric O2 promotes blooms of facultative anaerobes (eg. Proteobacteria) and restricts obligate anaerobes (eg. Firmicutes). Dysbiotic metabolites negatively affect host metabolism and immunity. Our novel compound (AuPhos) upregulates intestinal epithelial cell (IEC) mito function, attenuates colitis and corrects dysbiosis in humanized Il10-/- mice. We posit that AuPhos corrects IBD-associated dysbiotic metabolism. MethodsPrimary effect of AuPhos on mucosal Mito respiration and healing process was studied in ex vivo treated human colonic biopsies and piroxicam-accelerated (Px) Il10-/- mice. Secondary effect on microbiome was tested in DSS-colitis WT B6 and germ-free 129.SvEv WT or Il10-/- mice reconstituted with human IBD stool (Hu-Il10-/-). Mice were treated orally with AuPhos (10- or 25- mg/kg; q3d) or vehicle, stool samples collected for fecal lipocalin-2 (f-LCN2) assay and microbiome analyses using 16S rRNA sequencing. AuPhos effect on microbial metabolites was determined using untargeted global metabolomics. AuPhos-induced hypoxia in IECs was assessed by Hypoxyprobe-1 staining in sections from pimonidazole HCl-infused DSS-mice. Effect of AuPhos on enteric oxygenation was assessed by E. coli Nissle 1917 WT (aerobic respiration-proficient) and cytochrome oxidase (cydA) mutant (aerobic respiration-deficient). ResultsMetagenomic (16S) analysis revealed AuPhos reduced relative abundances of Proteobacteria and increased blooms of Firmicutes in uninflamed B6 WT, DSS-colitis, Hu-WT and Hu-Il10-/- mice. AuPhos also increased hypoxyprobe-1 staining in surface IECs suggesting enhanced O2 utilization. AuPhos-induced anaerobiosis was confirmed by a significant increase in cydA mutant compared to WT (O2-utlizing) E.coli. Ex vivo treatment of human biopsies with AuPhos showed significant increase in Mito mass, and complexes I and IV. Further, gene expression analysis of AuPhos-treated biopsies showed increase in stem cell markers (Lgr4, Lgr5, Lrig1), with concomitant decreases in pro-inflammatory markers (IL1{beta},MCP1, RankL). Histological investigation of AuPhos-fed Px-Il10-/- mice showed significantly decreased colitis score in AuPhos-treated Px-Il10-/- mice, with decrease in mRNA of pro-inflammatory cytokines and increase in Mito complexes (ND5, ATP6). AuPhos significantly altered microbial metabolites associated with SCFA synthesis, FAO, TCA cycle, tryptophan and polyamine biosynthesis pathways. AuPhos increased pyruvate, 4-hydroxybutyrate, 2-hydroxyglutarate and succinate, suggesting an upregulation of pyruvate and glutarate pathways of butyrate production. AuPhos reduced IBD-associated primary bile acids (BA) with concomitant increase in secondary BA (SBA). AuPhos treatment significantly decreased acylcarnitines and increased L-carnitine reflective of enhanced FAO. AuPhos increases TCA cycle intermediates and creatine, energy reservoir substrates indicating enhanced OxPHOS. Besides, AuPhos also upregulates tryptophan metabolism, decreases Kynurenine and its derivatives, and increases polyamine biosynthesis pathway (Putresceine and Spermine). ConclusionThese findings indicate that AuPhos-enhanced IEC mitochondrial function reduces enteric O2 delivery, which corrects disease-associated metabolomics by restoring short-chain fatty acids, SBA, AA and IEC energy metabolism. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/584471v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@41e437org.highwire.dtl.DTLVardef@1eddf37org.highwire.dtl.DTLVardef@1d9bff3org.highwire.dtl.DTLVardef@c925f0_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Colonic spermidine promotes the proliferation and migration of intestinal epithelial cells.

The gut microbiome, comprising trillions of diverse microorganisms, profoundly influences the efficient development and maintenance of the intestinal barrier. While shifts in microbial composition are observed in Inflammatory Bowel Disease (IBD) and Colorectal Cancer (CRC), the causal relationship between these changes and resolution of inflammation remains elusive. Notably, IBD is not only marked by shifts in microbial composition but also by changes in microbial metabolites. Polyamines, produced by both gut bacteria and human cells, have emerged as potential regulators of gut pathology, cancer and mucosal repair. Investigating how elevated polyamine levels influence intestinal epithelial cells (IEC) can provide insights into their role in the regeneration of mucosal epithelia and restoration of gut barrier functions. To dissect the complex interplay between the gut microbiome, polyamines, and IEC, we focused on the overrepresented bacterium B. uniformis and its primary metabolite, spermidine. Here, we show that B. uniformis, a dominant member of gut microbiota, expands during the repair & resolution phase of the chemically induced chronic murine colitis. Furthermore, we found that the abundance of colonic polyamines was also altered, with spermidine being the abundant polyamine. Our RNA sequencing and transcriptomic analysis of cultured colonic epithelial cells demonstrate that spermidine regulates the expression of genes and pathways involved in different cellular functions, including proliferation, differentiation, adhesion, lipid metabolism, migration, chemotaxis, and receptor expression. We also found that spermidine stimulates the proliferation of cultured colonic epithelial cells in vitro. Additionally, our findings indicate that spermidine enhances the migrations of enterocytes. Our study emphasizes the crucial functions of the gut microbiome and polyamines in governing the functions of intestinal epithelial cells. Thus, these microorganisms and their metabolic byproducts hold promise as prospective therapeutic agents.

microbiology↗

bioRxiv2023

AI-based antibody discovery platform identifies novel, diverse and pharmacologically active therapeutic antibodies against multiple SARS-CoV-2 strains

A critical aspect of a successful pandemic response is expedient antibody discovery, manufacturing and deployment of effective lifesaving treatments to patients around the world. However, typical drug discovery and development is a lengthy multi-step process that must align drug efficacy with multiple developability criteria and can take years to complete. In this context, artificial intelligence (AI), and especially machine learning (ML), have great potential to accelerate and improve the optimization of therapeutics, increasing their activity and safety as well as decreasing their development time and manufacturing costs. Here we present a novel, cost-effective and accelerated approach to therapeutic antibody discovery, that couples AI-designed human antibody libraries, biased for improved developability attributes with high throughput and sensitive screening technologies. The applicability of our platform for effective therapeutic antibody discovery is demonstrated here with the identification of a panel of human monoclonal antibodies that are novel, diverse and pharmacologically active. These first-generation antibodies, without the need for affinity maturation, bind to the SARS-CoV-2 spike protein with therapeutically-relevant specificity and affinity and display neutralization of SARS-CoV-2 viral infectivity across multiple strains. Altogether, this platform is well suited for rapid response to infectious threats, such as pandemic response. IMPORTANCEExpedient discovery and manufacturing of lifesaving therapeutics is critical for pandemic response. The recent COVID pandemic has highlighted the current inefficiencies and the need for improvements. To this end, we present our therapeutic antibody discovery platform that couples artificial intelligence (AI) and innovative high throughput technologies, and we demonstrate its applicability to rapid response. This platform enabled the isolation, characterization, and rapid identification of effective broadly neutralizing SARS-CoV-2 antibodies with good developability attributes, anticipated to fit our current process development and manufacturing platform. As such, this would benefit cost-of-goods and improve therapeutic access to patients. The AI-derived antibodies represent an advantageous therapeutic modality that can be developed and deployed fast, thus well suited for rapid response to infectious threats, such as pandemic response.

immunology↗

bioRxiv2023

Functional similarity of non-coding regions is revealed in phylogenetic average motif score representations

Here we frame the cis-regulatory code (that connects the regulatory functions of non-coding regions, such as promoters and UTRs, to their DNA sequences) as a representation building problem. Representation learning has emerged as a new approach to understand function of DNA and proteins, by projecting sequences into high-dimensional feature spaces, where the features are learned from data by a neural network. Inspired by these approaches, we seek to define a feature space where non-coding regions with similar regulatory functions are nearby each other. As a first attempt, we engineered features based on matches to biochemically characterized regulatory motifs in the DNA sequences of non-coding regions. Remarkably, we found that functionally similar promoters and 3 UTRs could be grouped together in a feature space defined by simple averages of the best match scores in (unaligned) orthologous non-coding regions, which we refer to as phylogenetic average motif scores. Perhaps most important, because this feature space is based on known motifs and not fit to any data, it is fully interpretable and not limited to any particular cell type or experimental context. We find that we can read off known regulatory relationships and evolutionary rewiring from visualizations of phylogenetic average motif score representations, and that predicted regulatory interactions based on neighbors in the feature space are borne out in transcription factor deletion experiments. Phylogenetic averages of match scores to known motifs is a baseline for representation learning applied to non-coding sequences, and may continue to improve as databases of motifs become more complete.

genomics↗

bioRxiv2022

A Legionella pneumophila effector impedes host gene silencing to promote virulence

RNA silencing is a gene silencing mechanism directed by small RNAs. Human miRNAs act as central regulators of host-bacteria interactions. However, it is unknown whether human pathogenic bacteria could impede RNA silencing to promote virulence. Here, we show that the Legionella pneumophila type IV-secreted effector LegK1 efficiently suppresses siRNA and miRNA activities in human cells. This effect depends on its known kinase activity, but also on its novel capacity, found here, to bind Argonaute (Ago) proteins. We further demonstrate that the ability of LegK1 to activate NF-{kappa}B signaling is required for RNA silencing suppression, establishing a link between effector-mediated NF-{kappa}B signaling and RNA silencing suppression. LegK1 also promotes L. pneumophila growth in both amoeba and human macrophages, supporting a role for this effector in virulence. Finally, we show that, in infected-macrophages, the latter activity relies, in part, on the genetic targeting of human Ago4. These findings indicate that a L. pneumophila effector has evolved to suppress RNA silencing to promote virulence. Significance StatementIt is now well established that mammalian viruses suppress RNAi to promote their replication in host cells. However, whether mammalian pathogenic bacteria use a similar virulence strategy remains unknown. Here, we show that the LegK1 effector from Legionella pneumophia, the causal agent of Legionnaires disease, efficiently suppresses RNAi in human cells. This effect depends on its ability to interact with Argonaute (Ago) proteins and to activate NF-{kappa}B signaling. In addition, LegK1 promotes virulence in infected-macrophages through the genetic targeting of human Ago4. Based on the lack of NF-{kappa}B-related factors in amoebae, and on the presence of canonical Ago proteins in these natural L. pneumophila hosts, we propose that the RNAi suppression activity of LegK1 represents its primary virulence function.

molecular biology↗

bioRxiv2021

Developmental Dynamics of RNA Translation in the Human Brain

The precise regulation of gene expression is fundamental to neurodevelopment, plasticity, and cognitive function. While several studies have deeply profiled mRNA dynamics in the developing human brain, there is a fundamental gap in our understanding of accompanying translational regulation. We perform ribosome profiling from more than 70 human prenatal and adult cortex samples across ontogeny and into adulthood, mapping translation events at nucleotide resolution. In addition to characterizing the translational regulation of annotated open reading frames (ORFs), we identify thousands of previously unknown translation events, including small open reading frames (sORFs) that give rise to human- and/or brain-specific microproteins, many of which we independently verify using size-selected proteomics. Ribosome profiling in stem cell-derived human neuronal cultures further corroborates these findings and shows that several neuronal activity-induced long non-coding RNAs (lncRNAs), including LINC00473, a primate-specific lncRNA implicated in depression, encode previously undescribed microproteins. Physicochemical analysis of these brain microproteinss identifies a large class harboring arginine-glycine-glycine (RGG) repeats as strong candidates for regulating RNA metabolism. Moreover, we find that, collectively, these previously unknown human brain sORFs are enriched for variants associated with schizophrenia. In addition to significantly expanding the translational landscape of the developing brain, this atlas will serve as a rich resource for the annotation and functional interrogation of thousands of previously unknown brain-specific protein products.

neuroscience↗

bioRxiv2021

Protein-lipid interaction at low pH induces oligomerisation of the MakA cytotoxin from Vibrio cholerae

Many pathogenic bacteria produce protein toxins that target and perturb host cell membranes. The secreted -pore-forming toxins (-PFTs) cause membrane damage via pore formation. This study demonstrates a remarkable, hitherto unknown mechanism by an -PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other -PFTs. In contrast, MakA protein alone induces tube-like structures in the acidic lysosomal host cell compartment. In vitro studies unravel the dynamics of tubular growth, which occur in a pH-, lipid- and concentration-dependent manner. A 3.7-[A] cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. In its active -PFT conformation, MakA embeds its transmembrane helices into a thin annular lipid bilayer and spirals around a central cavity. Our study provides molecular insights into a novel tubulation mechanism of an -PFT protein, revealing a new mode of action by a secreted bacterial toxin.

cell biology↗

bioRxiv2021

Structures of the human peroxisomal fatty acid transporter ABCD1 in a lipid environment

The peroxisomal very long chain fatty acid (VLCFA) transporter ABCD1 is central to cellular fatty acid catabolism and lipid biosynthesis. Its dysfunction underlies toxic cytosolic accumulation of VLCFAs, progressive nervous system demyelination, and neurological impairments including the potentially fatal disease X-linked adrenoleukodystrophy (X-ALD). Molecular details underlying substrate recognition and transport by ABCD1 are poorly understood. Here we determined cryo-EM structures of ABCD1 in phospholipid nanodiscs in a nucleotide bound conformation open to the peroxisomal lumen and an inward facing conformation open to the cytosol at up to 3.5 [A] resolution that reveal key details of its transmembrane cavity and ATP dependent conformational transitions. We identify structural elements distinguishing ABCD1 from its closest homologs and show that coenzyme A (CoA) esters of VLCFAs modulate ABCD1 activity in a species dependent manner. Together, our data support a transport mechanism where only the CoA moieties of VLCFA-CoAs enter the hydrophilic transmembrane cavity while the acyl chains extend out into the surrounding membrane bilayer, help rationalize disease causing mutations, and provide a framework for ABCD1 targeted structure-based drug design.

biochemistry↗

bioRxiv2021

How sequence context-dependent mutability drives mutation rate variation in the genome

The rate of mutations varies >100-fold across the genome, altering the rate of evolution, and susceptibility to genetic diseases. The strongest predictor of mutation rate is the sequence itself, varying 75-fold between trinucleotides. The fact that DNA sequence drives its own mutation rate raises a simple but important prediction; highly mutable sequences will mutate more frequently and eliminate themselves in favour of sequences with lower mutability, leading to a lower equilibrium mutation rate. However, purifying selection constrains changes in mutable sequences, causing higher rates of mutation. We conduct a simulation using real human mutation data to test if (1) DNA evolves to a low equilibrium mutation rate and (2) purifying selection causes a higher equilibrium mutation rate in the genomes most important regions. We explore how this simple process affects sequence evolution in the genome, and discuss the implications for modelling evolution and susceptibility to DNA damage.

evolutionary biology↗

bioRxiv2021

A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion

The protein MakA was discovered as a motility-associated secreted toxin from Vibrio cholerae, Here, we show that MakA is part of a gene cluster encoding four additional proteins: MakB, MakC, MakD and MakE. The MakA, MakB and MakE proteins were readily detected in culture supernatants of wild type V. cholerae whereas secretion was very much reduced from a flagellum deficient mutant. Crystal structures of MakA, MakB and MakE revealed structural relationship to a superfamily of bacterial pore-forming proteins. Cloning and expression of MakA/B/E in Escherichia coli resulted in toxicity of the bacteria towards Caenorhabditis elegans used as a predatory organism model. None of these Mak proteins alone or in pairwise combinations were cytolytic but an equimolar mixture of MakA, MakB and MakE acted as a tripartite cytolytic toxin in vitro causing lysis of erythrocytes and cytotoxicity on cultured human colon carcinoma cells. Formation of oligomeric complexes on liposomes was observed by electron microscopy. Oligomer interaction with membranes was initiated by MakA membrane binding followed by MakB and MakE joining in formation of a pore structure. A predicted membrane insertion domain of MakA was shown by site-directed mutagenesis to be essential for toxicity towards C. elegans. Bioinformatic analyses revealed that the makCDBAE gene cluster is present as a novel genomic island in the vast majority of sequenced genomes of V. cholerae and the fish pathogen V. anguillarum. We suggest that the hitherto unrecognized cytolytic MakA/B/E toxin can contribute to Vibrionaceae fitness and virulence potential in different host environments and organisms. Significance StatementVibrio cholerae, responsible for outbreaks and pandemics of cholera disease, is a highly motile organism by virtue of a single flagellum. We describe that the flagellum facilitates the secretion of three V. cholerae proteins encoded by a hitherto unrecognized novel genomic island. The proteins MakA/B/E can form a tripartite cytolytic toxin that lyses erythrocytes and is cytotoxic to cultured human cells. A structural basis for the Mak protein cytolytic activity was obtained by X-ray crystallography. Flagellum-facilitated secretion, remarkably ensuring spatially co-ordinated delivery of Mak proteins, revealed a new role for the V. cholerae flagellum considered of particular significance for the bacterial environmental persistence. Our findings will pave the way for the development of new diagnostics and therapeutic strategies against pathogenic Vibrionaceae.

biochemistry↗