Biology subjects

Rafiq, M.

Publications and source records attributed to Rafiq, M..

10 recordsLinked to original sources

bioRxiv2026

From Hummingbird to Elephant: Amyloid Formation in Natural Transthyretin Variants

Transthyretin (TTR) is a secreted protein associated with cardiac and other amyloid diseases via misfolding. We have previously shown that agitation of human TTR solutions at neutral pH results in aggregation and fibril formation. Here we report that agitation-induced aggregation of TTR from species with very different heart rates (Annas hummingbird, hbTTR, and African elephant, aeTTR) differs from that of human TTR (huTTR). Aggregation of hbTTR is slow and favors formation of smaller, fibrillar aggregates, while aeTTR aggregation is rapid and favors larger, more amorphous particles. Spherical, early-stage oligomeric intermediates were found for all variants by mass photometry and electron microscopy. The slow aggregation of hbTTR matches its resistance to denaturation by 8 M urea. The widely different aggregation behavior exhibited by these naturally occurring TTR variants in response to mechanical agitation under close to physiological conditions provides insight into how small sequence differences can contribute to the evolutionary fitness of different animals.

biophysics↗

bioRxiv2026

DM: a simple solution to suppress air-water interface interactions in cryo-EM

The air-water interface (AWI) remains the primary barrier to routine high-resolution cryo-EM structure determination, driving protein adsorption, structural denaturation, and restricted particle orientations during vitrification. Here, we describe a simple and broadly applicable strategy to mitigate these effects using the mild non-ionic detergent n-decyl-{beta}-D-maltopyranoside (DM). Addition of DM at low millimolar concentrations immediately prior to vitrification consistently suppresses AWI-driven artifacts, resulting in improved angular sampling, reduced structural damage, and enhanced reconstruction quality across diverse macromolecular systems. Using this approach, we obtained a high-resolution reconstruction of the 65 kDa Nucleophosmin 1 pentamer, a target previously limited by severe preferred orientation issues. We further show that DM promotes isotropic particle distributions for high-resolution reconstruction of hemagglutinin, transthyretin, as well as suppressing denaturation of aldolase while stabilizing its C-terminus. Our results indicate that DM effectively passivates deleterious air-water interface interactions without compromising particle integrity. These results establish DM as an effective additive for improving the robustness of single-particle cryo-EM sample preparation. O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/716008v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@108a6edorg.highwire.dtl.DTLVardef@10728b4org.highwire.dtl.DTLVardef@1014b2eorg.highwire.dtl.DTLVardef@1eed745_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Pharmacological Inhibition of SLC33A1 Promotes Endoplasmic Reticulum Hyperoxidation and Induces Adaptive IRE1/XBP1s Signaling

The endoplasmic reticulum (ER) transporter solute carrier family 33 member 1 (SLC33A1) has emerged as an attractive therapeutic target in etiologically diverse diseases, ranging from lung cancer to neurodegenerative disorders. Yet, no pharmacologic SLC33A1 modulators have been described. Here, we show that the small molecule IXA4, a highly selective activator of the adaptive IRE1/XBP1s signaling arm of the unfolded protein response (UPR), binds to SLC33A1 and inhibits its activity. Genetic depletion of SLC33A1 phenocopies the selective induction of IRE1/XBP1s signaling brought about by IXA4 treatment. Chemoproteomic analyses and cryo-electron microscopy show that IXA4 binds SLC33A1 within the central channel to inhibit transport of its substrate metabolite(s). Binding of IXA4 to SLC33A1 leads to the accumulation of oxidized glutathione within the ER, hyperoxidizing the ER lumen and inducing activation of adaptive IRE1/XBP1s signaling. Consistent with this function, we find that pharmacologic inhibition of SLC33A1 with IXA4 selectively reduces viability of KEAP1-deficient lung adenocarcinoma cells that have elevated levels of glutathione, mimicking the sensitivity of these cells to genetic deletion of SLC33A1. Our work demonstrates a new physiologic role of SLC33A1 in regulation of ER redox homeostasis and designates IXA4 as a pharmacologic inhibitor of SLC33A1 that can be used to evaluate the biological impact and therapeutic utility of SLC33A1 inhibition in homeostasis and in disease.

cell biology↗

bioRxiv2025

Convergent evolution of a fungal effector enabling phagosome membrane penetration

The ability of pathogens to evade phagosomal killing is critical for their pathogenicity. Previously, we had identified the HscA effector protein in the clinically important fungal pathogen Aspergillus fumigatus, which redirects conidia-containing phagosomes from the degradative to the non-degradative pathway. Here, we discovered a pathogenic form of this surface protein, determined by a single tyrosine residue (Y) at position 596, which is lacking in most fungi analyzed, that have a leucine (L) instead. Y596 enables HscA to penetrate the phagosomal membrane. In line, the introduction of a single L- to-Y exchange in the orthologous Ssb protein of Saccharomyces cerevisiae enabled the protein to penetrate phagosomal membranes that was reduced by deletion of one of the two Y-encoding SSB genes in the pathogenic fungus Candida glabrata. These data suggest a convergent evolution of HscA/Ssb proteins among human-pathogenic fungi and that a single amino acid exchange determines a virulence factor. HighlightsO_LIHscA with Y596 penetrates host cell membranes and causes phagosomal damage C_LIO_LIMost fungi (96%) have a leucine at the respective position C_LIO_LIAn L-to-Y mutation in Ssb enables S. cerevisiae to damage phagosomes C_LIO_LIRecruitment of ESCRT complex to the phagosomal membrane requires human p11 protein C_LI

microbiology↗

bioRxiv2023

Computational characterization of the interactions between novel Boronic Acid derivatives with urokinase-type plasminogen activator (uPA) and their binding energy

1.Urokinase type plasminogen activator is expected to play a significant role in metastasis therefore various inhibitors are being prepared for this target protein. However, the binding site with residues that are involved in binding and inhibition is unidentified. Hence, comprehensive computational techniques are applied for finding the binding pocket, important amino acid residues and for the characterization of the binding energy of the best ligand among seven novel boronic acid derivative inhibitors within the binding pocket. Among seven test compounds, C14H21BN2O2S showed best results in structure based molecular docking through Molecular Operating Environment (MOE) and GOLD suit with -3.2481 kcal/mol binding affinity and 46.4523 GOLD Score. C14H21BN2O2S also showed high binding affinity within the binding pocket in DFT (Density Functional Theory) studies. DFT was carried out using hybrid functional B3LYP in combination with basis set LANL2DZ level of density functional theory on the extracted geometry of bound ligand C14H21BN2O2S to the binding pocket of uPA with a -2 charge on amino-acid residue ASP189. Computational analysis values on Geometric Optimization (opt), Single Point Energy (SPE) and Self-Consistent Reaction Field (SCRF) were 53.9, -66.3 and -49.0 respectively. Hence it is concluded that C14H21BN2O2S shows better binding with uPA binding pocket when there is a negative two charge on it ASP189 amino acid residue in the binding pocket. These seven ligands were also used for generating pharmacophore model through random selection with genetic algorithm by MOE having sensitivity of 79% towards the test set, specificity of 78% towards test set and 51% calculated Matthews coefficient correlation. 2. Author SummaryBoronic-acid based proteasome inhibitor like Bortezomib and Ixazomib are Food and Drug Administration (FDA) approved drugs, which are being used for fighting cancer. They can be considered as a template for understanding the pharmacokinetics and role of Boronic-acid ligands in the process. Boron-based warheads with stabilised functionality along with reduced toxicity are beneficent therapeutically. We have utilized computational quantum mechanical techniques in predicting binding free energies for ligands and proteins in a solvent environment. Instead of providing precise estimations, these techniques are more suitable for prediction purposes. The main challenge is developing inhibitors for uPA sub-sites that have high selectivity, potency, and improved pharmacokinetic properties. We have used Molecular docking and ligand-based techniques to analyze the binding interactions between seven ligands and uPA. Among these ligands, C14H21BN2O2S is identified as the most appropriate inhibitor based on scores and its interactions with specific receptor amino acid residues. Computational quantum mechanical studies are conducted using electron density and hybrid functional B3LYP to determine the binding energy. A pharmacophore model is designed to identify crucial descriptors and search for compounds that can effectively inhibit uPA. The models accuracy is assessed through QSAR analysis, which reveals favorable hydrogen bond donor and acceptor groups as well as aromatic hydrophobic rings in proximity to the ligands. The designed model demonstrates good sensitivity, specificity, and calculated Matthews coefficient correlation.

bioinformatics↗

bioRxiv2022

Re-direction of phagosomes to the recycling expulsion pathway by a fungal pathogen

The analysis of host-pathogen interactions bears the potential to discover novel pathogenicity mechanisms and to obtain novel insights into basic mechanisms of cell biology. Here, we obtained unprecedented insight into both. We discovered that the HscA protein on the conidial surface of the clinically important human-pathogenic fungus Aspergillus fumigatus acts as an effector protein. It inhibits phagosome maturation and reprograms phagosomes for expulsion of conidia. HscA anchors the human p11 protein to phagosomes. p11 is a decisive factor for targeting phagosomes either to the degradative or secretory pathway. The relevance of our findings is indicated by the identification of an SNP in the non-coding region of the human p11 gene that affects its translation and is associated with heightened susceptibility to invasive pulmonary aspergillosis.

cell biology↗

bioRxiv2021

PLB-985 neutrophil-like cells release antifungal extracellular vesicles against the human-pathogenic fungus Aspergillus fumigatus

Fungal infections remain a major global concern. Emerging fungal pathogens and increasing rates of resistance mean that additional research efforts and resources must be allocated to advancing our understanding of fungal pathogenesis and developing new therapeutic interventions. Neutrophilic granulocytes are a major cell type involved in protection against the important fungal pathogen Aspergillus fumigatus, where they employ numerous defense mechanisms, including production of antimicrobial extracellular vesicles. A major draw-back to work with neutrophils is the lack of a suitable cell line system for the study of fungal pathogenesis. To address this problem, we assessed the feasibility of using differentiated PLB-985 neutrophil-like cells as an in vitro model to study A. fumigatus infection. We find that dimethylformamide-differentiated PLB-985 cells provide a useful recapitulation of many aspects of A. fumigatus interactions with primary human polymorphonuclear leukocytes. We show that differentiated PLB-985 cells phagocytose fungal conidia and acidify conidia-containing phagolysosomes similar to primary neutrophils, release neutrophil extracellular traps, and also produce antifungal extracellular vesicles in response to infection. In addition, we provide an improved method for the isolation of extracellular vesicles produced during infection by employing a size-exclusion chromatography-based approach. Advanced LC-MS/MS proteomics revealed an enrichment of extracellular vesicle marker proteins and a decrease of cytoplasmic proteins in extracellular vesicles isolated using this improved method. Ultimately, we find that differentiated PLB-985 cells can serve as a genetically tractable model to study many aspects of A. fumigatus pathogenesis. IMPORTANCEPolymorphonuclear leukocytes are an important defense against human fungal pathogens, yet our model systems to study this group of cells remains very limited in scope. In this study, we established that differentiated PLB-985 cells can serve as a model to recapitulate several important aspects of human polymorphonuclear leukocyte interactions with the important human fungal pathogen Aspergillus fumigatus. The proposed addition of a cultured neutrophil-like cell line to the experimental toolbox to study fungal pathogenesis will allow for a more mechanistic description of neutrophil antifungal biology. In addition, the easier handling of the cell line compared to primary human neutrophils allowed us to use PLB-985 cells to provide an improved method for isolation of neutrophil-derived extracellular vesicles using size-exclusion chromatography. Together, these results provide significant tools and a baseline knowledge for the future study of neutrophil-derived extracellular vesicles in the laboratory.

microbiology↗

bioRxiv2020

Language network connectivity increases in prodromal Alzheimer's disease

Language production deficits occur early in the course of Alzheimers disease (AD); however, only few studies have focused on language functional networks in prodromal AD. The current study aims to uncover the extent of language alteration at a prodromal stage, on a behavioral, structural and functional level, using univariate and multivariate analyses. Twenty-four AD participants and 24 matched healthy controls underwent a comprehensive language evaluation, a structural T1-3D MRI and resting-state fMRI. We performed seed-based analyses, using the left inferior frontal gyrus and left posterior temporal gyrus as seeds. Then, we analyzed connectivity between executive control networks and language network in each group. Finally, we used multivariate pattern analyses to test whether the two groups could be distinguished based on the pattern of atrophy within the language network; atrophy within the executive control networks, as well as the pattern of functional connectivity within the language network; and functional connectivity within executive control networks. AD participants had language impairment during standardized language tasks and connected-speech production. Univariate analyses were not able to discriminate participants at this stage, while multivariate pattern analyses could significantly predict the group membership of prodromal patients and healthy controls, both when classifying atrophy patterns or connectivity patterns of the language network. Language functional networks could discriminate AD participants better than executive control networks. Most notably, they revealed an increased connectivity at a prodromal stage. Multivariate analyses represent a useful tool for investigating the functional and structural (re-)organization of the neural bases of language. HighlightsLanguage network connectivity discriminates prodromal AD from healthy controls Language network connectivity increases in prodromal AD Atrophy patterns in the language network do not correlate with connectivity patterns in AD

neuroscience↗

bioRxiv2020

Transcription Factors STAT5A and SPI1 Reveals RHBDD2 as a Potential Biomarker in Sepsis and Septic Shock

Sepsis is a serious health situation caused by uncontrolled infection and septic shock is a severe condition of sepsis. RHBDD2 is a member of the rhomboid superfamily which is overexpressed in different types of cancer and associated with ER stress and estrogen receptor. Using microarray gene expression data and using different computational techniques this study investigated the role of RHBDD2 in sepsis and septic shock. Finds functional annotation of RHBDD2 using co-expression analysis and identified the deregulation of RHBDD2 in sepsis using differential expression analysis. Results show that RHBDD2 is overexpressed in sepsis and septic shock. The GO enrichment analysis, KEGG pathways, and biological functions of the RHBDD2 co-expressed genes module show that it is involved in most of the sepsis-related biological functions and also plays a role in most of the infection-related pathways which lead to sepsis and septic shock. RHBDD2 is regulated by STAT5A and SPI1 transcription factors in sepsis and septic shock. The identification of the RHBDD2 as a biomarker may facilitate in septic shock diagnosis, treatment, and prognosis.

bioinformatics↗

bioRxiv2020

The dynamic surface proteomes of allergenic fungal conidia

Fungal spores and hyphal fragments play an important role as allergens in respiratory diseases. In this study, we performed trypsin shaving and secretome analyses to identify the surface-exposed proteins and secreted/shed proteins of Aspergillus fumigatus conidia, respectively. We investigated the surface proteome under different conditions, including temperature variation and germination. We found that the surface proteome of resting A. fumigatus conidia is not static, but instead unexpectedly dynamic, as evidenced by drastically different surface proteomes under different growth conditions. Knockouts of two abundant A. fumigatus surface proteins, ScwA and CweA, were found to function only in fine-tuning the cell wall stress response, implying that the conidial surface is very robust against perturbations. We then compared the surface proteome of A. fumigatus to other allergy-inducing molds, including Alternaria alternata, Penicillium rubens, and Cladosporium herbarum, and performed comparative proteomics on resting and swollen conidia, as well as secreted proteins from germinating conidia. We detected 125 protein ortholog groups, including 80 with putative catalytic activity, in the extracellular region of all four molds, and 42 nonorthologous proteins produced solely by A. fumigatus. Ultimately, this study highlights the dynamic nature of the A. fumigatus conidial surface and provides targets for future diagnostics and immunotherapy.

microbiology↗