Biology subjects

Sultana, A.

Publications and source records attributed to Sultana, A..

7 recordsLinked to original sources

bioRxiv2024

Coevolving Residues Distant from the Ligand Binding Site are Involved in GAF Domain Function

Ligand binding to GAF domains regulates the activity of associated catalytic domains in a wide variety of proteins. For instance, cGMP binding to the GAFa domain of phosphodiesterase 5 (PDE5) activates the cGMP-hydrolyzing catalytic domain in the protein. However, the residues involved and the mechanism of GAF domain function are not entirely clear. Here, combining computational and experimental analysis, we show that two highly coevolving residues distant from the ligand binding site play a critical role in GAF domain allostery. Specifically, Statistical Coupling Analysis (SCA) of GAF domain sequences revealed the highest coevolution score for residues L267 and F295. Molecular dynamics (MD) simulations of both apo and holo forms of the wild type and mutant (L267A and F295A) PDE5 GAFa domains revealed significant alterations in structural dynamics and interaction with cGMP. Incorporation of the mutations in a Bioluminescence Resonance Energy Transfer (BRET)-based biosensor, which reports a ligand-induced conformational change, revealed a change in the conformation of the GAF domain and an increase in the EC50 of cGMP-induced conformational change. Similar results were obtained regarding cGMP-induced conformational change in the full-length PDE5 and in the fluorescence of the GAF domain fluorescent protein, miRFP670nano3. Finally, structural analysis of conformers observed in MD simulations revealed a possible mechanism underlying the impact of mutations of these two coevolving residues in the PDE5 GAFa domain. Our results provide insight into the role of distant, coevolving residues in GAF domain allostery, and may aid in understanding evolution of allostery in proteins.

molecular biology↗

bioRxiv2023

PERK-mediated induction of miR-5p and miR-3p arms of miR-616 regulates cell growth by targeting c-MYC

C/EBP homologous protein (CHOP), also known as DNA damage-inducible transcript 3 (DDIT3), is a member of CCAAT/enhancer-binding protein (C/EBP) family. The expression of CHOP is upregulated during unfolded protein response (UPR), and sustained CHOP activity plays an important role in UPR-induced apoptosis. MicroRNA-616 is localized in an intron of the CHOP gene. However, regulation of miR-616 expression during UPR and its function in breast cancer is not clearly understood. We show that miR-5p/-3p arms of miR-616 are expressed with levels of 5p arm higher than 3p arm. During conditions of UPR, the expression of miR-5p and miR-3p arms of miR-616 and its host gene (CHOP) was concordantly increased primarily in a PERK-dependent manner. We show that ectopic expression of miR-616 significantly suppressed cell growth and colony formation, whereas knockout of miR-616 increased it. We identified that MYC proto-oncogene (c-MYC) gene is repressed during the UPR and targeted by miR-616. Further, we show that expression of miR-616 and CHOP is downregulated in human breast cancer, where expression of miR-616 was associated with poor overall survival (OS) in luminal A subtype and better OS HER2 subtype of breast cancer. In summary, our results suggest a dual function for the DDIT3 locus, where CHOP protein and miR-616 can co-operate to regulate cancer progression.

cancer biology↗

bioRxiv2023

A Slow but Steady NanoLuc: R162A mutation results in a decreased, but stable, NanoLuc activity

NanoLuc (NLuc) luciferase has found extensive application in designing a range of biological assays including gene expression analysis, protein-protein interaction and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remains elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable bioluminescence activity of NLuc in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the absence and presence of the ligand. Further, analysis of trajectories for hydrogen bonds (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in in vitro assays performed with furimazine. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity.

biophysics↗

bioRxiv2022

Antibody avidity and multi-specificity combined to confer protection against SARS-CoV-2 and resilience against viral escape

SARS-CoV-2, the causative agent of COVID-19, has been responsible for a global pandemic. Monoclonal antibodies have been used as antiviral therapeutics, but have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs), and in deployment by the need for high doses. In this study, we leverage the MULTI-specific, multi-Affinity antiBODY (Multabody, MB) platform, derived from the human apoferritin protomer, to drive the multimerization of antibody fragments and generate exceptionally potent and broad SARS-CoV-2 neutralizers. CryoEM revealed a high degree of homogeneity for the core of these engineered antibody-like molecules at 2.1 [A] resolution. We demonstrate that neutralization potency improvements of the MB over corresponding IgGs translates into superior in vivo protection: in the SARS-CoV-2 mouse challenge model, comparable in vivo protection was achieved for the MB delivered at 30x lower dose compared to the corresponding IgGs. Furthermore, we show how MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding IgGs lose their ability to neutralize potently. Multiple mAb specificities could also be combined into a single MB molecule to expand the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.

immunology↗

bioRxiv2022

An in vivo and in silico evaluation of the magical hepatoprotective potentialities of Gynura procumbens: a promising agent for combating hepatotoxicity

IntroductionLiver being the most important metabolic organ of the body performs a wide variety of vital functions. Hepatic cell injury occurs by the activation of reactive oxygen species (ROS) by CCl4, xenobiotics and other toxic substances generated through cytochrome P450 dependent step resulting from covalent bond formation with lipoproteins and nucleic acids. Observing the alarming state of hepatotoxic patients worldwide, different medicinal plants and their properties can be explored to combat against such free radical degermation of liver. This paper evaluates the antioxidant property of Gynura procumbens in both in silico and in an in vivo assay, and its hepatoprotective activity in CCl4 induced hepatotoxicity. Materials and MethodsGynura procumbens leaves were collected and extracted using 50% ethanol. Required chemicals (CCl4), standard drug (Silymarin) and blood serum analyzing kits were stocked. The in vivo tests were performed in 140 healthy Wister albino male rats under well controlled parameters dividing into 14 groups, strictly maintaining IEAC protocols. In silico molecular docking and ADMET studies were performed and the results were analyzed statistically. Results and discussionThe body weight increased significantly in CCl4 induced, G. procumbens administered hepatotoxic rats. The increase in SGPT, SGOT, ALP, creatinine, LFH, triglycerides, LDL, SOD, MDA, total cholesterol, DNA fragmentation ranges, {gamma}GT levels of CCl4 treated group was decreased by both standard drug Silymarin and G. procumbens leaf extract. On the other hand, G. procumbens increased HDL levels and displayed contrasting results in CAT level tests. Some results contradicted with the negative controlled group displaying varying efficacy between leaf extract and Silymarin. In the molecular docking analysis, G. procumbens phytoconstituents performed poorly against TGF-{beta}1 compared to the control drug Galunisertib while 26 phytoconstituents scored better than the control, bezafibrate against PPAR-. Flavonoids and phenolic compounds performed better than other constituents in providing hepatoprotective activity.

bioinformatics↗

bioRxiv2022

Optimization of conditions for conjugation of outer membrane vesicles of Salmonella Typhimurium with oral delivery systems using response surface method

BackgroundOuter membrane vesicles (OMVs) released from Salmonella Typhimurium can become the effective vaccine candidate. For efficient delivery through oral route, the OMVs are conjugated with some delivery systems. The current study was aimed at optimization of conditions required for conjugation of OMVs with nano- or microparticles for maximum entrapment of OMV in terms of protein concentration. MethodsThe OMVs of Salmonella Typhimurium were conjugated under three optimum conditions of pH, temperature and ratio (nanoparticles or microparticles: OMVs) predicted by response surface method. The efficiency of conjugation was determined by entrapment of OMVs in nano/microparticles. ResultThe pH and temperature were not influential conditions in case of conjugation of OMVs with chitosan nanoparticles (Ch-NP) and poly-lactide co-glycolide microparticles (PLG-MP) while they had influence in case of poly(anhydride) nanoparticles. In case of Ch-NP and PLG-MP, the optimum ratio for maximum entrapment was found to be 1:10 and 1:9 respectively. The optimum pH and temperature was found to be 7 and 24{degrees}C respectively for conjugation of poly(anhydride) nanoparticles. The optimized conditions did not alter the protein profile and immunogenic potential of conjugated vaccines.

biochemistry↗

bioRxiv2020

Inhibition of ITK differentiates GVT and GVHD in allo-HSCT

Allogeneic hematopoietic stem cell transplantation is a life-saving treatment for many malignant and nonmalignant diseases. Donor T cells contained within the graft prevent tumor recurrence via graft-versus-tumor (GVT) effects, however, also cause graft-versus-host disease (GVHD). Novel treatment strategies are therefore needed to allow maintenance of GVT while suppressing GVHD. Here we show using murine models, that targeting IL-2-inducible T cell kinase (ITK) in donor T cells reduces GVHD while preserving the beneficial GVT effects. Donor T cells from Itk-/- mice exhibit significantly reduced production of inflammatory cytokines and migration to GVHD target organs such as liver and small intestine, while maintaining GVT efficacy against primary B-ALL tumors. Itk-/- T cells exhibited reduced expression of IRF4 and decreased JAK/STAT signaling activity, but preserved cytotoxicity, which was accompanied by upregulation of Eomesodermin (Eomes), which was necessary for GVT function. A novel peptide inhibitor ITK signaling is also able to prevent GVHD. This novel peptide inhibitor also reduced cytokine production in mice and human T cells. Altogether, our data suggest that inhibiting ITK could be a therapeutic strategy to reduce GVHD while preserving the beneficial GVT effects following allo-HSCT treatment. Key PointsO_LIInhibiting ITK by a novel peptide significantly reduces GVHD but retains GVT. C_LIO_LIITK deficient donor T cells exhibit minimal GVHD, but maintain GVT activity. C_LIO_LIITK deficient donor T cells exhibit significantly reduced production of inflammatory cytokines and migration to GVHD target organs. C_LIO_LIEomes is required for GVT effect. C_LI

immunology↗