Biology subjects

Rahman, K.

Publications and source records attributed to Rahman, K..

12 recordsLinked to original sources

bioRxiv2025

IFITM1 and IFITM3 cooperate to restrict virus entry in endolysosomes

Interferon-induced transmembrane (IFITM) proteins are potent innate immune factors that restrict an array of viruses at the entry stage of infection. We previously characterized a GxxxG motif in the CD225 domain of human IFITM3 that mediates its multimerization, which is essential for the reduction of membrane fluidity by IFITM3 and for its antiviral activity against Influenza A virus. Here, using an unbiased approach coupling immunoprecipitation with mass spectrometry, we show that the GxxxG motif is also important for the interaction of IFITM3 with other proteins, including IFITM1. While IFITM1 is primarily regarded as a cell surface protein that restricts the entry of viruses fusing at the plasma membrane, this model is based mostly on overexpression studies and is at odds with some studies showing that it can restrict endocytic viruses. Here, we show that endogenous IFITM1 and IFITM3 co-reside in membranes of acidic late endosomes and lysosomes (endolysosomes) and form a protein-protein complex as determined by co-immunoprecipitation and proximity ligation assay. Knockdown of endogenous IFITM3 resulted in enhanced localization of IFITM1 at the plasma membrane, indicating that IFITM3 promotes IFITM1 localization to endolysosomes. To assess the antiviral protection conferred by endogenous IFITM1 and IFITM3 against viruses fusing at endolysosomal membranes, we measured cell entry mediated by the Influenza A fusogen hemagglutinin (HA). While knockdown of IFITM3 significantly boosted HA-mediated entry, combined knockdown of both IFITM3 and IFITM1 boosted entry even further. These results suggest that endogenous IFITM1 restricts Influenza A virus entry in a manner that is non-redundant with IFITM3, and that IFITM1 and IFITM3 inhibit virus entry in a cooperative manner.

microbiology↗

bioRxiv2024

SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

The CD225/Dispanins superfamily consists of membrane proteins that regulate vesicular transport and membrane fusion events driving neurotransmission, glucose transport, and antiviral immunity. However, how the CD225 domain controls membrane trafficking was unknown. We reveal that the CD225 domain contains a SNARE-like motif that enables interaction with cellular SNARE fusogens. Proline rich transmembrane protein 2 (PRRT2) encodes a SNARE-like motif that enables interaction with neuronal SNARE proteins, and mutations therein disrupt SNARE binding and are linked to neurological disease. Another CD225 member, interferon-induced transmembrane protein 3 (IFITM3), protects cells against Influenza A virus infection. IFITM3 interacts with SNARE proteins that mediate late endosome-late endosome (homotypic) fusion and late endosome-lysosome (heterotypic) fusion. IFITM3 binds to syntaxin 7 (STX7) in cells and in vitro, and mutations that abrogate STX7 binding cause loss of antiviral activity against Influenza A virus. Mechanistically, IFITM3 disrupts assembly of the SNARE complex controlling homotypic fusion and accelerates the trafficking of endosomal cargo to lysosomes. Our results suggest that SNARE modulation plays a previously unrecognized role in the diverse functions performed by CD225 proteins.

cell biology↗

bioRxiv2024

Detection of Potential Phytochemicals against ctxAB Toxin to Combat Cholera

Vibrio cholerae is a gram-negative curved rod-shaped bacterium responsible for cholera, an intestinal infection characterized by severe acute watery diarrhea that can be fatal if untreated. The major virulence factor for Vibrio cholerae is cholera toxin (ctx), a potent toxin which has two subunits, A and B (ctxAB), that are crucial for the progression of the deadly disease. The B subunit is a pentavalent protein that binds to the intestinal mucosa to allow internalization of the A subunit to initiate rice-watery diarrhea. This study explored potential phytochemicals to inhibit the B subunit from binding to the host mucosa by examining their interaction with ctxB. Proteins encoded by the three genotypes of the ctxB gene (ctxB1, ctxB3, and ctxB7) present in the El Tor biotype strains of V. cholerae O1 responsible for the currently ongoing seventh cholera pandemic were targeted. Analysis of 52 phytochemicals obtained from PubChem identified a group of phytochemicals based on their binding affinity scores with the proteins of ctxB genotypes. Assessment of drug-likeliness and toxicity risk highlighted two phytochemicals, Limonin and Emodin, as the best potential candidates for inhibiting the mode of action of cholera toxin. Molecular dynamic (MD) simulation-based MM-PBSA analysis suggested these compounds stably interact with the receptors. Molecular docking and molecular dynamic simulations revealed that these phytochemicals might have potential to inhibit the cholera toxin function and reduce the severity of cholera. Author SummaryCholera toxin (ctx) subunits A and B (ctxAB) play a significant role in the disease, with the B subunit facilitating the binding of the toxin to the intestinal cells, and the A subunit entering the cells, resulting in the cellular changes that lead to watery diarrhea. To inhibit the B subunit of cholera toxin from binding host mucosa, we explored candidate phytochemicals instead of synthetic molecules as drug agents by examining their binding behavior with ctxB using a molecular docking approach. The molecular docking, molecular dynamic simulations, and toxicity analyses identified two phytochemical compounds that have the potential to inhibit the mechanism of cholera toxin.

bioinformatics↗

bioRxiv2023

Alternative splicing expands the antiviral IFITM repertoire in Chinese horseshoe bats

The interferon response is shaped by the evolutionary arms race between hosts and the pathogens they carry. The human interferon-induced transmembrane protein (IFITM) family consists of three antiviral IFITM genes that arose by gene duplication, they restrict virus entry and are key players of the interferon response. Yet, little is known about IFITMs in other mammals. Here, we identified an IFITM gene in Chinese horseshoe bat, a natural host of SARS-coronaviruses, that is alternatively spliced to produce two IFITM isoforms. These bat IFITMs have conserved structures in vitro and differential antiviral activities against influenza A virus and coronaviruses including SARS- and MERS-coronavirus. In parallel with human IFITM1-3, the bat IFITM isoforms localize to distinct cellular compartments. Further analysis of IFITM repertoires in 205 mammals reveals that alternative splicing is a ubiquitous strategy for IFITM diversification, albeit less widely adopted than gene duplication. These findings showcase an example of convergent evolution where species-specific selection pressures led to expansion of the IFITM family through multiple means, underscoring the importance of IFITM diversity as a component of innate immunity.

microbiology↗

bioRxiv2022

Requirement for specific bacterial genome maintenance pathways in repair of C8-linked pyrrolobenzodiazepine (PBD) bi-aryl monomer-mediated DNA damage

Pyrrolobenzodiazepines (PBDs) are naturally occurring DNA binding compounds that possess anti-tumor and anti-bacterial activity. Chemical modifications of PBDs can result in improved DNA binding, sequence specificity and enhanced efficacy. More recently, synthetic PBD monomers have shown promise as payloads for antibody drug conjugates and antibacterial agents. The precise mechanism of action of these PBD monomers and their role in causing DNA damage remains to be elucidated. Here we characterized the damage-inducing potential of two C8-linked PBD bi-aryl monomers in Caulobacter crescentus and investigated the strategies employed by cells to repair the same. We show that these compounds cause DNA damage and efficiently kill bacteria, in a manner comparable to the extensively used DNA cross-linking agent mitomycin-C (MMC). However, in stark contrast to MMC which employs a mutagenic lesion tolerance pathway, we implicate essential functions for error-free mechanisms in repairing PBD monomer-mediated damage. We find that survival is severely compromised in cells lacking nucleotide excision repair and to a lesser extent, in cells with impaired recombination-based repair. Loss of nucleotide excision repair leads to significant increase in double-strand breaks, underscoring the critical role of this pathway in mediating repair of PBD-induced DNA lesions. Together, our study provides comprehensive insights into how mono-alkylating DNA-targeting therapeutic compounds like PBD monomers challenge cell growth, and identifies the specific mechanisms employed by the cell to counter the same.

microbiology↗

bioRxiv2022

Cholesterol binds the amphipathic helix of IFITM3 and regulates antiviral activity

The interferon-induced transmembrane (IFITM) proteins broadly inhibit the entry of diverse pathogenic viruses, including Influenza A virus (IAV), Zika virus, HIV-1, and SARS coronaviruses by inhibiting virus-cell membrane fusion. IFITM3 was previously shown to disrupt cholesterol trafficking, but the functional relationship between IFITM3 and cholesterol remains unclear. We previously showed that inhibition of IAV entry by IFITM3 is associated with its ability to promote cellular membrane rigidity, and these activities are functionally linked by a shared requirement for the amphipathic helix (AH) found in the intramembrane domain (IMD) of IFITM3. Furthermore, it has been shown that the AH of IFITM3 alters lipid membranes in vitro in a cholesterol-dependent manner. Therefore, we aimed to elucidate the relationship between IFITM3 and cholesterol in more detail. Using a fluorescence-based in vitro binding assay, we found that a peptide derived from the AH of IFITM3 directly interacted with the cholesterol analog, NBD-cholesterol, while other regions of the IFITM3 IMD did not, and native cholesterol competed with this interaction. In addition, recombinant full-length IFITM3 protein also exhibited NBD-cholesterol binding activity. Importantly, previously characterized mutations within the AH of IFITM3 that strongly inhibit antiviral function (F63Q and F67Q) disrupted AH structure in solution, inhibited cholesterol binding in vitro, and restricted bilayer insertion in silico. Our data suggest that direct interactions with cholesterol may contribute to the inhibition of membrane fusion pore formation by IFITM3. These findings may facilitate the design of therapeutic peptides for use in broad-spectrum antiviral therapy.

immunology↗

bioRxiv2021

A calibrated cell-based functional assay to aide classification of MLH1 DNA mismatch repair gene variants

PURPOSEFunctional assays provide important evidence for classifying the disease significance of germline variants in the DNA mismatch repair genes. We sought to develop a cell-based approach for testing the function of variants of uncertain significance (VUS) in the MLH1 gene. METHODSUsing CRISPR gene editing, we knocked-in MLH1 VUS into the endogenous MLH1 loci in human embryonic stem cells. We examined their impact at the RNA and protein level, including their ability to maintain stability of microsatellite sequences and instigate a DNA damage response. We calibrated these assays by testing well-established pathogenic and benign control variants. RESULTSFive VUS resulted in functionally abnormal protein, 15 VUS resulted in functionally normal protein, and one VUS showed mixed results. Furthermore, we converted the functional outputs into a single odds in favor of pathogenicity score for each VUS. CONCLUSIONOur CRISPR-based functional assay successfully models phenotypes observed in patients in a cellular context. Using this approach, we generated evidence for or against pathogenicity for utilization by variant classification expert panels. Ultimately, this information will assist in proper diagnosis and disease management for suspected Lynch syndrome patients.

cancer biology↗

bioRxiv2020

Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to its oligomeric state

The interferon-inducible transmembrane (IFITM) proteins belong to the Dispanin/CD225 family and inhibit diverse virus infections. IFITM3 reduces membrane fusion between cells and virions through a poorly characterized mechanism. We identified a GxxxG motif in many CD225 proteins, including IFITM3 and proline rich transmembrane protein 2 (PRRT2). Mutation of PRRT2, a regulator of neurotransmitter release, at glycine-305 was previously linked to paroxysmal neurological disorders in humans. Here, we show that glycine-305 and the homologous site in IFITM3, glycine-95, drive protein oligomerization from within a GxxxG motif. Mutation of glycine-95 in IFITM3 disrupted its oligomerization and reduced its antiviral activity against Influenza A and HIV-1. The oligomerization-defective variant was used to reveal that IFITM3 promotes membrane rigidity in a glycine-95-dependent manner. Furthermore, a compound which counteracts virus inhibition by IFITM3, amphotericin B, prevented the IFITM3-mediated rigidification of membranes. Overall, these data suggest that IFITM3 oligomers inhibit virus-cell fusion by promoting membrane rigidity.

evolutionary biology↗

bioRxiv2020

Reprogramming the endogenous type III-A CRISPR-Cas system for genome editing, RNA interference and CRISPRi screening in Mycobacterium tuberculosis

Mycobacterium tuberculosis (M.tb) causes the current leading infectious disease. Examination of the functional genomics of M.tb and development of drugs and vaccines are hampered by the complicated and time-consuming genetic manipulation techniques for M.tb. Here, we reprogrammed M.tb endogenous type III-A CRISPR-Cas10 system for simple and efficient gene editing, RNA interference and screening via simple delivery of a plasmid harboring a mini-CRISPR array, thereby avoiding the introduction of exogenous proteins and minimizing proteotoxicity. We demonstrated that M.tb genes were efficiently and specifically knocked-in/out by this system, which was confirmed by whole-genome sequencing. This system was further employed for single and simultaneous multiple-gene RNA interference. Moreover, we successfully applied this system for genome-wide CRISPR interference screening to identify the in-vitro and intracellular growth-regulating genes. This system can be extensively used to explore the functional genomics of M.tb and facilitate the development of new anti-Mycobacterial drugs and vaccines. SummaryTuberculosis caused by Mycobacterium tuberculosis (M.tb) is the current leading infectious disease affecting more than ten million people annually. To dissect the functional genomics and understand its virulence, persistence, and antibiotics resistance, a powerful genome editing tool and high-throughput screening methods are desperately wanted. Our study developed an efficient and a robust tool for genome editing and RNA interference in M.tb using its endogenous CRISPR cas10 system. Moreover, the system has been successfully applied for genome-wide CRISPR interference screening. This tool could be employed to explore the functional genomics of M.tb and facilitate the development of anti-M.tb drugs and vaccines.

microbiology↗

bioRxiv2019

Delineating the Organization of Projection Neuron Subsets with Multi-fluorescent Rabies Virus Tracing Tool

The elegant functions of the brain are facilitated by sophisticated connections between neurons, the architecture of which is frequently characterized by one nucleus connecting to multiple targets via projection neurons. Delineating the sub-nucleus fine architecture of projection neurons in a certain nucleus could greatly facilitate its circuit, computational, and functional resolution. Here, we developed multi-fluorescent rabies virus to delineate the fine organization of corticothalamic projection neuron subsets in the primary visual cortex (V1). By simultaneously labeling multiple distinct subsets of corticothalamic projection neurons in V1 from their target nuclei in thalamus (dLGN, LP, LD), we observed that V1-dLGN corticothalamic neurons were densely concentrated in layer VI, except for several sparsely scattered neurons in layer V, while V1-LP and V1-LD corticothalamic neurons were localized to both layers V and VI. Meanwhile, we observed a fraction of V1 corticothalamic neurons targeting multiple thalamic nuclei, which was further confirmed by fMOST whole-brain imaging. We further conceptually proposed an upgraded sub-nucleus tracing system with higher throughput (21 subsets) for more complex architectural tracing. The multi-fluorescent RV tracing tool can be extensively applied to resolve architecture of projection neuron subsets, with a strong potential to delineate the computational and functional organization of these nuclei.

neuroscience↗

bioRxiv2019

A glycogenin homolog controls Toxoplasma gondii growth via glycosylation of an E3 ubiquitin ligase

Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is uniquely modified in protists by an O2-dependent prolyl hydroxylase that generates the attachment site for a defined pentasaccharide. Previous studies demonstrated the importance of the core glycan for growth of the parasite Toxoplasma gondii in fibroblasts, but the significance of the non-reducing terminal sugar was unknown. Here, we find that a homolog of glycogenin, an enzyme that can initiate and prime glycogen synthesis in yeast and animals, is required to catalyze the addition of an -galactose in 3-linkage to the subterminal glucose to complete pentasaccharide assembly in cells. A strong selectivity of the enzyme (Gat1) for Skp1 in extracts is consistent with other evidence that Skp1 is the sole target of the glycosyltransferase pathway. gat1-disruption results in slow growth attesting to the importance of the terminal sugar. Molecular dynamics simulations provide an explanation for this finding and confirm the potential of the full glycan to control Skp1 organization as in the amoebozoan Dictyostelium despite the different terminal disaccharide assembled by different glycosyltransferases. Though Gat1 also exhibits low -glucosyltransferase activity like glycogenin, autoglycosylation is not detected and gat1-disruption reveals no effect on starch accumulation. A crystal structure of the ortholog from the crop pathogen Pythium ultimum explains the distinct substrate preference and regiospecificity relative to glycogenin. A phylogenetic analysis suggests that Gat1 is related to the evolutionary progenitor of glycogenin, and acquired a role in glycogen formation following the ancestral disappearance of the underlying Skp1 glycosyltransferase prior to amoebozoan emergence.

microbiology↗

bioRxiv2019

A quantitative medico-botanical expedition of Fairy Meadows National Park, Diamir, Gilgit Baltistan, Pakistan

This study was conducted to investigate the ethnobotanical knowledge of the population of the Fairy Meadow National Park, Diamir, Gilgit Baltistan. The study area was previously ignored due to physical barriers, remoteness and religious extremism. The use of medicinal plants for various maladies, known to the elders of the community and passed orally to the younger generation was documented. A total of 146 informants were interviewed using semi-structured questionnaires. The data was quantitatively analyzed employing frequency of citation (FC), use value (UV), relative frequency of citation (RFC) along with Pearson correlation coefficient (PCC). A total of 90 plants species belonging to 77 genera and 49 different families have been documented. These medicinal plants were used against 55 diseases, especially stomach problems (23.3%), cough (17.7%), asthma and fever (16.6%). For 31 out of 90 plants species which had been reported either, new uses (28 plants) or new use-report (3 plants) were found. New reported medicinal plants include Allium gilgitensis, Astragalus gilgitensis and Pedicularia flava. The majority of the documented plants were wild collected (86%), herbs (60%), and leaves were the most widely used part (27%). The common method of preparation was powder (27%) mainly administered orally (81.7%). The highest use values were found for Berberis lyceum (5.47), Thymus lineari s(5.07) and Rhododendron anthopogon (5.0), while the plants with greater relative frequency of citation were Berberis lyceum (0.97), Thymus linearis (0.89) and Rhododendron anthopogon (0.75). The Pearson correlation coefficient is 0.836 between RFC and UV showing high positive association. This study was an extension to the ethnobotanical work done in Pakistan previously. We documented a wealth of traditional knowledge, and could record the uses of various species for the first time from Pakistan. The new use reports and new plants reported supplement the foundation of pharmacology and new drug development for complex and challenging disease

plant biology↗